Fatty acid degradation in plants.

Prog. Lipid Res. Vol. 31, No. 4, pp. 417-446, 1992 Printed in Great Britain. All rights reserved

0163-7827/92/$15.00 © 1992 Pergamon Press Ltd

FATTY ACID D E G R A D A T I O N IN PLANTS BERNTGERHARDT Institut fiir Botanik, Universitdt M6nster, Schlossgarten 3, D - 4 4 ~ M6nster, Germany

CONTENTS I. II. III. IV.

INTRODUCTION PHYSIOLOGICALROLE SUnCELLULARLOCALIZATION PEROXISOMALft.-OXIDATIONSYSTEMOF HIOX-~.RPLANTS A. Degradation of common straight-chain fatty acids 1. Properties of fl-oxidation enzymes (a) Acyl-CoA synthetase (EC 6.2.1.3) (b) Acyl-CoA oxidase (EC 1.1.3) (c) Multifunctionai protein (d) Thiolase (EC 2.3.1.16) (e) A2, A3-Enoyl-CoA isomerase (EC 5.3.3.8) (f) D-3-Hydroxyacyl-CoA dehydratase (g) 2,4-Dienoyl-CoA reductase (EC 1.3.1) 2. Overall fl-oxidation (a) Saturated fatty acids (b) Unsaturated fatty acids B. Degradation of ricinoleic acid C. Degradation of propionyl-CoA D. Degradation of branched-chain 2-oxo acids I. Activation by oxidative decarboxylation 2. Pathways of degradation (a) Degradation of 2-oxo-3-methylvalerate (b) Degradation of 2-oxoisovalerate (c) Degradation of 2-oxoisocaproate E. Particularities of the peroxisomal fatty acid degrading system F. Fate of acetyl-CoA G. Energetic aspects H. Transport of fatty acids I. Regulation V. MITOCHONDRIALft-OxIDATIONIN H i o ~ PLA~rs A. Carnitine acyltransferase B. Paimitoylcarnitine-dependent oxygen uptake

C. Enzymes of fl-oxidation D. Conclusion VI. -OXIDATIONOF FATTYACIDS VII. DEGRADATIONOF FATTY ALCOHOLS VIII. FATTY ACID DEGRADATION IN ALGA~ ACKNOWLE~E~nBNT

REFERENCES

417 418 418 419 419 419 421 422 423 423 423 424 424 425 425 426 428 430 431 432 432 432 432 432 434 435 436 436 437 438 438 439 44O 441 441 442 442 443 443

I. I N T R O D U C T I O N

Fatty acid degradation is an oxidative process. There are several oxidative processes acting on fatty acids in plants: ~t-oxidation,/~-oxidation, in-chain oxidation and w-oxidation. This review will cover only ~t-oxidation and r-oxidation. The r-oxidation pathway comprising repetitive r-oxidations including several modifications of r-oxidation, is the sole process currently known to lead to complete degradation of fatty acids. In contrast, ~t-oxidation appears to be restricted to long-chain fatty acids and to shorten them not beyond Cj2 chain length, if at all acting repeatedly on a long-chain fatty acid. In-chain oxidation and oJ-oxidation generate hydroxy, oxo and epoxy fatty acids and are involved in the formation of polyfunctional fatty acids, constituents of surface lipid polymers (cutin, suberin). In-chain oxidation is also involved in the synthesis of ricinoleic acid (o-12hydroxyoleic acid), a major component of the storage triacylglycerols of castor bean seeds, and presumably involved in the synthesis of other hydroxy and oxo fatty acids contained 417

418

B. GERHARDT

in certain storage lipids. As both in-chain oxidation and co-oxidation are processes which fulfil biosynthetic rather than catabolic functions they are not included in this account. The lipoxygenase reaction which acts on fatty acids possessing a 1-cis,4-cis-pentadiene structure like linoleic and linolenic acid, and the metabolism of its reaction products have been the subject of some recent reviews accentuating different aspects. I-3 Therefore, the lipoxygenase reaction will not be covered in this review. The status of research on fatty acid degradation in plants prior to 1980 has been reviewed by Galliard. 4 Since then, significant progress in this area of research has been made due to renewed interest in the topic. Focussing on certain aspects, advances in our understanding of fatty acid degradation in plants have been treated in recent reviews. ~ The objectives of this article are to survey current knowledge about the biochemistry of plant systems degrading physiologically relevant fatty acids, and to delineate the integration of fatty acid degradation into plant cell metabolism. Data resulting from studies on fatty acid degradation in heterotrophic organisms are only integrated when needed in context. At any level, comparison of fatty acid degradation in plants with that in heterotrophic organisms is not intended. Reviews on fl-oxidation in mammalian cells have been published very recently. 9'1° Fatty acid degradation in algae is considered in a separate section of this review (Section VIII). Thus, the review is restricted to higher plants in all of its other sections. II. P H Y S I O L O G I C A L R O L E

Fatty acid catabolism is a major metabolic pathway in lipid-storing tissues of oilseeds when the lipid reserves are mobilized to sustain growth of the seedling during germination. The fatty acids released from the storage triacylglycerols (primarily) do not serve as respiratory substrates. Following degradation of the fatty acids to their constituent acctyl-units the carbon of the fatty acids is channelled into gluconeogenesis (sucrose synthesis) via the glyoxylate cycle. 1~'1sUtilization of reserve lipids as respiratory substrates has been demonstrated based on the ~3C/~SCratio of respired CO s in the inflorescence of Philodendron selloum K.Koch (Araceae) ~3 and suggested to occur at the very early germination stage of oilseeds. ~4'~5Whether the fatty acids serve directly or indirectly as respiratory substrates is unknown. Fatty acid degradation in plants is not confined to lipid-storing tissues. Many, if not all, plant tissues have at least the capacity for fatty acid degradation by fl-oxidation (Section III). In the majority of plant tissues, fatty acid degradation is certainly only a minor metabolic process. Nevertheless, this process is essential considering the harmful properties of free fatty acids which can originate from both protein and membrane lipid turnover. Contribution of fatty acids to respiration in mature plant tissues is poorly documented. 16 Initial wound respiration of certain bulky storage organs (e.g. potato (Solanum tuberosum L.) tubers) is caused by oxidation of fatty acids originating from cellular membrane degradation induced by wounding. 17-~9Fatty acids released at membrane breakdown also appear to form the respiratory substrate in tissue cultures at sucrose starvation, s° In contrast, carbon of fatty acids originating from membrane degradation in senescing leaves is thought to be directed into gluconeogenesis and to be saved for growing parts of the plant. 2~-s3Significant respiration of valine which is catabolized via acyl-CoA intermediates has been demonstrated in pea (Pisum sativum L.) roots, s4 III. S U B C E L L U L A R L O C A L I Z A T I O N

Within the higher plant cell, fatty acid degradation is located in a distinct subcellular compartment, the peroxisome. 5'6"sPeroxisomes are regular constituents of the higher plant cell. 25'26They can be subcategorized into glyoxysomes and nongiyoxysomal peroxisomes with respect to metabolism of acetyl-CoA generated at fl-oxidation (Section IV.F)) Glyoxysomes are characteristic of lipid-mobilizing tissues of oilseeds and are defined by housing the glyoxylate cycle directing acetyl-CoA into gluconeogenesis. ~2Following their

Fatty acid degradationin plants

419

discovery in 1967,27 glyoxysomes of the castor bean endosperm were shown to carry out fl-oxidation. 2s Localization of fl-oxidation in glyoxysomes is very well established today. 25,26 Nonglyoxysomal peroxisomes occurring in the majority of higher plant tissues lack glyoxylate cycle activity and are known to be involved in certain tissue-specific activities (e.g. leaf peroxisomes are involved in photorespiration). Ability of nonglyoxysomal peroxisomes to perform //-oxidation was first reported in 1981/83. 29-32 Localization of //-oxidation in nonglyoxysomal peroxisomes has been demonstrated for photosynthetic tissue, roots and other plant tissues/organs, devoid of storage lipids, of various plant species (see Ref. 5; Table 2). Where examined so far, //-oxidation enzymes and/or //-oxidation activity have been demonstrated in nonglyoxysomal peroxisomes. Thus, both glyoxysomes and nonglyoxysomal peroxisomes are able to perform//-oxidation. This led to the concept 5'8 that (i) enzymes of//-oxidation are regular constituents of higher plant peroxisomes, (ii) fatty acid catabolism by//-oxidation is a basic function of higher plant peroxisomes, and (iii) higher plant peroxisomes are competent in degrading physiologically relevant fatty acids of different molecular structure. As peroxisomes are regular constituents of higher plant cells, capacity for//-oxidation appears to exist in most, if not all, higher plant cells. Whether higher plant cells possess a mitochondrial fatty acid degrading system in addition to the//-oxidation system located in peroxisomes is a matter of debate at present (Section V). The u-oxidation process (Section VI) is thought to be localized at the endoplasmic reticulum. However, experimental evidence for that is scarce. IV. PEROXISOMAL/~-OXIDATION SYSTEM OF HIGHER PLANTS Studies on the biochemistry of the peroxisomal//-oxidation system in higher plant cells have been performed using glyoxysomes from lipid-mobilizing tissues of various oilseeds and nonglyoxysomal peroxisomes isolated mainly from mung bean (Vigna radiata L.) hypocotyls and potato tubers (selected since they are standard objects of research on plant mitochondria). Since there are no qualitative differences between the results obtained with either of the peroxisome type, it will not consequently be indicated whether data presented refer to experiments performed with glyoxysomes or nonglyoxysomal peroxisomes.

A. Degradation of Common Straight-Chain Fatty Acids This section covers degradation of straight-chain saturated and unsaturated fatty acids (Cn, n > 3) carrying no additional functional group(s). Following activation by acyl-CoA synthetase saturated fatty acids are degraded by repetitive passages through the fl-oxidation reaction sequence. This includes the enzymes acyl-CoA oxidase, multifunctional protein and thiolase. The enzymes catalyze first oxidation, hydration and second oxidation, and thiolytic cleavage, respectively, of acyl-CoA. As the cis double bond(s) of physiologically relevant unsaturated fatty acids form a barrier to degradation by continuous passages through the //-oxidation reaction sequence, additional enzymes (A2,A3-enoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, D-3-hydroxyacyl-CoA dehydratase) are involved in degradation of activated unsaturated fatty acids.

1. Properties of fl-Oxidation Enzymes Enzymes involved in peroxisomal degradation of common, straight-chain saturated and unsaturated fatty acids have been purified only from cucumber (Cucumis sativus L.) cotyledons)3-38However, acyl-CoA synthetase and 2,4-dienoyl-CoA reductase have not yet been purified from a plant source. Molecular and kinetic properties of the purified enzymes are shown in Table 1. Kinetic properties of floxidation enzymes have also been studied using broken peroxisomes as the enzyme source. Published data on fl-oxidation enzyme kinetics would allow the calculation of first order rate constants as indicators of substrate

100 (butyryl-CoA) 20 (palmitoyl-CoA)

K. 0m)

tSubstrate used in the assay.

*Ref. 34.

Enzymes were purified from cucumber cotyledons.

pHOpfimum

8.34.5

160

0.45 (palmitoyl-CoA)t

Specific activity (pkat mg -I)

V,m (pkat mg -I)

10 (crotonyl-CoA) Hydratase act.: Dehydrogenase act. 50:1" (C4 substrates) 3"1 10:1 (C~0 substrates)

7.8

IEP

41

24

(2-trans -hexenoyl-CoA)

13

(crotonyi-CoA)

34

140 (crotonyl-CoA) 182 29 (2-trans -hexenoyl-CoA)

Monomer

Monomer

Homodimer

Grade of oligomerization 9.8*

76,500

74,000

Isoform II

Isoform I

150,000

33

Size, M r

Oxidasc

Multifunctional protein 35

9 4

8.1 0.69 (3-trans -dccenoyl-CoA)

8.5 0.07 (acetoacctyl-CoA)

8.0

Homodimer

Homodimer

Homodimer

9.0

8.7 (3-c/~-hexenoyl-CoA)

170 (3-c/s -hexenoybCoA)

65,000

50,000

90,000

7.5-8.0

7.0 (2-trans -decenoyl-CoA)

(2-trans -butenoyl-CoA)

1.5

(2-trans -decenoyl-CoA)

9.5

(2-trans-butenoyl-CoA )

110

(DL-3-hydroxydecanoyl-CoA)

Dehydratasc 3g

lsomerase 37

Thiolase36

TABLE 1. Properties of Purified Enzymes involved in Peroxisomai Fatty Acid Degradation

4~ FO C,

Fatty acid degradation in plants T~

421

2. Relative Activities of ~-Oxidation Enzymes in Glyoxysomes (A) and Nonglyoxysomal Peroxisomes (B) Activity related to acyl-CoA oxidase activity Multifunctional Protein

Peroxisomes isolated from (A) Cucumis sativus L., cotyledons Ricinus communis L., endosperm Helianthus annuus L., cotyledons (B) Spinacia oleracea L., leaves Pisum sativum L., leaves Vigna radiata L., hypocotyls Zea mays L., seedling roots Solarium tuberosum L., tubers Pisum sativum L., cotyledons Persea americana Mill, mesocarp Aram maculatam L., spadix appendices

Aeyl-CoA synthetase

Aeyl-CoA oxidase

Enoyl-CoA hydratase

3-OH-AcyiCoA DH

Thiolase

Ref.

1

27

0.7

0.03

6

0.5

1 (3.3)*

15

0.8

1 (7.7)

64

0.2

1 (0.6)

0.3

1 (0.7)

1.0

1 (I.0)

1.3 0.1

1

41a

8.4

0.3

5

8.3

0.5

0.5

5

9.6

0.7

0.3

5

37

2.5

2.2

5

I (0.4)

40

1.8

0.5

5

1 (!.4)

16

1.5

0.2

30

2.5

0.3

31

8.5

0.3

1 (0.4)

2.8

0.2

1 (3.9)

52

0.5

1 (1.7)

75

15

16

4.7

AcyI-CoA synthetase and aeyl-CoA oxidase activities assayed with C,csubstrates. Activities of multifunctional protein and thiolase assayed with C4-substrates. *The values in brackets give the absolute activity (nkat (mg organdie protein) -~) of acyl-CoA oxidase.

specificities. However, it is difficult to assess whether presuppositions required for such calculations have been fulfilled experimentally. Critical micellar concentrations of substrates in particular have hardly been considered in previous publications (this aspect is also relevant with respect to studies on relative chain length specificities of t-oxidation enzymes). Therefore, first order rate constants are not calculated from kinetic data. Data on the activity of the enzymes catalyzing the basic reactions of t-oxidation are given in Table 2. The enzymes of fatty acid degradation in higher plant peroxisomes appear to be soluble matrix proteins or to be loosely associated with the organelle membrane,34, 36,39-41 except acyl-CoA synthetase which is tightly bound to the organelle membrane. 4°

(a) Acyl-CoA synthetase (EC 6.2.1.3. Common, straight-chain fatty acids are activated to their corresponding acyl-CoAs in a reaction requiring ATP, Mg 2+ and CoASH. 4°' 42 Besides acyl-CoA, AMP and pyrophosphate have been demonstrated as reaction products which were formed in a 1:1:1 stoichiometry. 4° Cofactor requirements and product analysis indicate that the fatty acids are activated by an acyl-CoA synthetase. Pyrophosphatase activity has not been detected in peroxisomes (Ref. 41 a, Fischer and Gerhardt, unpublished data). Studies on the substrate specificity of acyl-CoA synthetase indicated that short-chain (n ,: 8) fatty acids are poor substrates for the enzyme. 4°' 42 The acyl-CoA synthetase of mung bean hypocotyl peroxisomes showed also low activity ( < 3 0 % of the activity observed with palmitate as substrate) towards medium-chain fatty acids. The enzyme activated preferentially the CIs unsaturated fatty acids. 4° However, low or no activity of acyl-CoA syntbetases towards oleic acid has also been reported? 3 Apparent Vm~ and Km values for Ci0:0 to Ci6:0 fatty acids have been determined for glyoxysomal acyl-CoA synthetases? 3 Both Vm~ and Km values peaked mainly for C12:0and/or C~4:0fatty acids. The Km values were mainly in the range of 10-100 #M. With respect to palmitate, Km values

422

B. GERHARDT

between 3 and 700 #M have been reported for acyl-CoA synthetase from different plant sources.40, 43

Results on the substrate specificity of acyl-CoA synthetase suggest that the substrate specificity determined in vitro does not necessarily mirror the in vivo specificity of the enzyme which, in the cases of oilseeds, can be deduced from the fatty acid moiety of the storage triacylglycerols. For example, the acyl-CoA synthetase of glyoxysomes isolated from cotyledons of a rape (Brassica napus L.) variety containing predominantly erucic and oleic acid in its reserve lipids, did not activate these fatty acids in vitro. 43 But a low in vitro activity of acyl-CoA synthetase towards fatty acids which are predominant components of the storage triacylglycerols of a tissue may be in fact a sufficient activity in vivo if related to flux rates. However, flux rates through fatty acid degradation pathways are unknown, except in the castor bean endosperm (Section IV.I). The substrate specificity of acyl-CoA synthetase is not restricted to physiological requirements. It has been shown that mung bean hypocotyl peroxisomes and pea leaf extracts (their acyl-CoA synthetase activity is assumed to be primarily due to the peroxisomal enzyme) activate ricinoleate with appreciable rates. 44'45 However, ricinoleic acid is certainly not a constituent of the lipids occurring in mung bean hypocotyls or pea leaves. Acyl-CoA synthetase appears to be an enzyme tightly bound to the peroxisome membrane?° Following membrane fractionation, acyl-CoA synthetase activity was recovered in the fraction containing very hydrophobic proteins. As the enzyme showed latency and resistance to protease (thermolysin) treatment when breakage of peroxisomes from mung bean hypocotyls,4° pea leaves4° or sunflower (Helianthus annuus L.) cotyledons (B/inning and Gerhardt, unpublished data) was avoided at activity assay, the enzyme or at least its essential domain(s) seem to be located on the matrix face of the organdie membrane. (b) Acyl-CoA oxidase (EC 1.1.3). The first oxidation step of the peroxisomal fl-oxidation reaction sequence, i.e. oxidation of acyl-CoA to 2-trans-enoyl-CoA is catalyzed by an acyl-CoA oxidase. The reaction requires molecular oxygen28 which serves as the acceptor of the two electrons liberated at acyl-CoA oxidation. The electron transfer from acyl-CoA to oxygen does not require any cofactor other than that bound to the enzyme as a prosthetic group. Molecular oxygen is reduced to H20228"32 which in turn is apparently degraded by catalase within peroxisomes. Oxygen uptake and H202 and enoyl-CoA formation occur in a 1:1:1 stoichiometry.32 Formation of enoyl-CoA was demonstrated using a reconstituted fl-oxidation system to which the remaining enzymes needed for NADH formation at fl-oxidation were added as purified enzymes of animal sources. 32 As the enzymes added exhibit known specificity (3-hydroxyacyl-CoA dehydrogenase acts specifically on the L-isomer46 generated only from 2-trans-enoyl-CoA by enoyl-CoA hydratase) the enoyl-CoA formed by plant acyl-CoA oxidas¢ has indirectly been demonstrated to be the 2-trans isomer. Formation of 2-trans-enoyl-CoA at the acyl-CoA oxidase reaction has been demonstrated directly for the reaction of rat liver peroxisomes catalyzed by acyl-CoA oxidase.47 The plant acyl-CoA oxidase is believed to be a FAD-containing protein like the acyl-CoA oxidase of mammalian~ and yeast49 peroxisomes. The amino acid composition of the acyl-CoA oxidase purified from cucumber cotyledons has been analyzed.33 Acyl-CoA oxidase acts preferentially on acyl-CoAs of Ct2 to Cj6 chain length) 3' 43. 50 Activity towards butyryl-CoA higher than that towards medium- or long-chain acyl-CoAs has been observed for acyl-CoA oxidase from some nonglyoxysomal peroxisomes. 3°' 3J, 5o However, the Km value for the enzyme of mung bean hypocotyl peroxisomes for butyryl-CoA was considerably higher than that for lauroyl-CoA or myristoyl-CoA. Low Kmvalues of acyl-CoA oxidase for Ct2 to Ct6 acyl-CoAs oxidized with high rates have been reported in some33'50but not in all cases. 43Thus, the data currently available on the kinetic properties of plant acyl-CoA oxidases suggest that activity and affinity of the enzyme towards acyl-CoAs of different chain length do not always change in parallel (in vitro). The

Fatty acid degradation in plants

423

Kmvalue of acyl-CoA oxidase for palmitoyl-CoA has been reported mainly in the range of 5--25 #M. (c) Multifunctionalprotein. In peroxisomal p-oxidation, a single protein catalyzes the hydration of 2-trans-enoyl-CoA to 3-hydroxyacyl-CoA and the subsequent oxidation of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA.34 As L-3-hydroxyacyl-CoA is exclusively formed from 2-trans-enoyl-CoA by mammalian mitochondrial enoyl-CoA hydratase it is reasonable to assume that in the course of peroxisomal ~-oxidation the L-isomer of 3-hydroxyacyl-CoA is a product of the enoyl-CoA hydratase (EC 4.2.1.17) activity as well as a substrate of the 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activity of the peroxisomal multifunctional protein. Based on sequence analysis of the cDNA of the multifunctional protein of rat liver peroxisomes it has been suggested that enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity of the protein are located on its N-terminal and C-terminal domains, respectively. 51 Besides the two mentioned activities, the multifunctional protein exhibits some D-3hydroxyacyl-CoA dehydratase activity. 35 The function of this activity has not yet been established as peroxisomes contain an individual protein carrying considerably higher D-3-hydroxyacyl-CoA dehydratase activity (Section IV.A.I.f). D-3-Hydroxyacyl-CoA is formed by the 2-enoyl-CoA hydratase activity of the multifunctional protein when 2-c/s-enoyl-CoA is the substrate instead of 2-trans-enoyl-CoA. Two peroxisomal isoforms of the multifunctional protein have been purified from cucumber cotyledons (Ref. 35; Table 1). The multifunctional protein uses NAD as electron acceptor at the oxidative reaction catalyzed. The Vmax values of enoyl-CoA hydratase activity of glyoxysomes from cotton (Gossypium hirsutum L.) cotyledons have been reported to decrease 100-fold as the chain length of the enoyl-CoA substrate increased from C4 to C~6.52The Km values increased from 0.2 to 3.0/zM over the chain length range studied. The continuous decrease in activity obtained with carbon chain length increase is in contrast to the observation that substrates of medium and/or long carbon chain are in general better substrates for p-oxidation enzymes than short-chain substrates. Available data on the substrate specificity of the enoyl-CoA hydratase activity of the purified multifunctional protein of cucumber cotyledon glyoxysomes are listed in Table I. (d) Thiolase (EC 2.3.1.16). The thiolytic cleavage of 3-oxoacyl-CoA (Cn) to acyl-CoA (Cn_ 2) and acetyl-CoA is catalyzed by a thiolase which has not yet been characterized with respect to its kinetic properties.

(e) A2,A3-Enoyl-CoAisomerase (EC 5.3.3.8). Degradation, by the ~-oxidation pathway, of a straight-chain unsaturated fatty acid with c/s double bond(s) extending from an odd-numbered carbon atom yields a 3-cis-enoyl-CoA intermediate when the position of the double bond has been reached. 3-Cis-enoyl-CoA is converted into 2-trans-enoyl CoA, a substrate of the multifunctional protein, by a A2,A3-enoyl-CoA isomerase. The reaction is reversible. 37The isomerase purified from cucumber cotyledons did not accept 2-cis-enoylCoA, 4-cis-enoylCoA or 2-trans,4-cis-dienoyl-CoA (the substrate of 2,4-dienoyI-CoA reductase; Section IV.A. 1.g) as substrate when the isomerase activity was assayed in the reverse direction. 37 Beside 3-cis-enoyl-CoA, 3-trans-enoyl-CoA was also converted to 2-trans-enoyl-CoA)7 However, the rate observed with 3-cis-hexenoyl-CoA as substrate was approximately 30 times higher than that observed with the substrate 3-trans-hexenoyl-CoA. When the chain length of the 3-trans-enoyl-CoA was increased, the rate observed with 3-trans-dodecenoylCoA as substrate was comparable to that obtained with the substrate 3-cis-hexenoyl-CoA. The result suggests that the activity of the isomerase is stimulated by both the c/s configuration of the substrate and elongation of the carbon chain (within a certain range of carbon chain length). The isomerase exhibited considerable substrate inhibition with all CoA esters accepted as substrate. 37

424

B. GERHARDT

(f) D-3-Hydroxyacyl-CoA dehydratase. A cis double bond extending from an even-numbered carbon atom of an unsaturated fatty acid forms a barrier to continuous degradation of the fatty acid by t-oxidation. The 2-cis-enoyl-CoA intermediate generated at the point of the double bond will be hydrated by the enoyl-CoA hydratase activity of the multifunctional protein, yielding D-3-hydroxyacyl-CoA instead of L-3-hydroxyacyl-CoA. However, the o-3-hydroxyacyl-CoA cannot be metabolized by the L-3-hydroxy-acyl-CoA dehydrogenase activity of the multifunctional protein. An epimerase converting O-3hydroxyacyl-CoA into L-3-hydroxyacyl-CoA was thought to be involved into t-oxidation at this point. However, it has very recently been demonstrated that a distinct 3-hydroxyacyl-CoA epimerase does not exist. 9'53 In peroxisomes, the epimerase activity results from two distinct reactions: dehydration of D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA by a novel o-3-hydroxyacyl-CoA dehydratase and subsequent hydration of the 2-trans-enoylCoA to L-3-hydroxyacyl-CoA by the enoyl-CoA hydratase activity of the multifunctional protein.3g, 53 Using enzymes of known specificity for product identification in/n vitro reconstitution experiments it has been demonstrated that the D-3-hydroxyacyl-CoA dehydratase purified from cucumber cotyledons converted D-3-hydroxyacyl(decanoyl)-CoA to 2-trans-enoyl(decenoyl)-CoA in a reversible reaction. 38 L-3-Hydroxyacyl(decanoyl)-CoA or 2-cisenoyl(decenoyl)-CoA were not accepted as substrates by the novel enzyme. Both activity and affinity of the enzyme seem to increase when the chain length of the substrate is increased (Table 1). Two isoforms of the novel D-3-hydroxyacyl-CoA dehydratase have been purified from cucumber cotyledons.38 The isoforms exhibited very similar, if not mainly identical, molecular and kinetic properties. The isoforms are thought to represent together the monofunctional "epimerase" protein described earlier for glyoxysomes of cucumber cotyledonsfl The "epimerase" protein carried the predominant proportion of the overall "epimerase" activity of the organelles, but some "epimerase" activity was also associated with both isoforms of the multifunctional protein isolated from cucumber cotyledons.35 Thus, besides the novel D-3-hydroxyacyl-CoA dehydratase, the multifunctional protein appears to carry some D-3-hydroxyacyl-CoA dehydratase activity. (g) 2,4-Dienoyl-CoA reductase (EC 1.3.1). Mammalian mitochondria do not possess D-3-hydroxyacyl-CoA dehydratase in order to circumvent the //-oxidation barrier(s) caused by cis double bond(s) extending from an even-numbered carbon atom of straight-chain unsaturated fatty acids. Also, D-3-hydroxyacyl-CoA dehydratase is probably involved in degradation of such fatty acids only to a minor extent in mammalian peroxisomes.9 The t-oxidation barrier considered here is circumvented in mammalian mitochondria and probably predominantly in mammalian peroxisomes by a 2,4-dienoylCoA reductase. 9' ~ Degradation, by t-oxidation, of straight-chain unsaturated fatty acids containing cis double bond(s) extending from an even-numbered carbon atom leads, at one point, to the formation of a 4-cis-enoyl-CoA intermediate. Oxidation of this intermediate by acyl-CoA oxidase yields 2-trans,4-cis-dienoyl-CoA. Hydration of 2-trans,4-cis-dienoyl-CoA by the multifunctional protein is considered to be a thermodynamically unfavorable reaction (Ref. 55; but see Section IV.A.2.b). It has been demonstrated that the 2-trans,4-cis-dienoylCoA intermediate is metabolized to 3-trans-enoyl-CoA (which in turn is substrate for the isomerase) in a NADPH-dependent reaction. The enzyme 2,4-dienoyl-CoA reductase catalyzing this reaction has been purified from mammalian mitochondria56 and peroxisomes)7 Using 2-trans,4-trans-decadienoyl.CoA as substrate (as the animal 2,4dienoyl-CoA reductase reduces both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA), 2,4dienoyl-CoA reductase activity has been demonstrated in glyoxysomes from cucumber cotyledonsfl Occurrence of 2,4-dienoyl-CoA reductase activity (assayed with sorbyl-CoA as substrate) has also been reported for peroxisomal fractions from pineapple (Ananas comosus Merr.) fruit tissue. 5s So far, 2,4-dienoyl-CoA reductase has neither been purified from a plant source nor been characterized using plant peroxisomes as the enzyme source.


425

2. Overall//-Oxidation Higher plant peroxisomes isolated from different tissues and various species catalyze both formation of NADH and acetyl-CoA in an hypotonic medium when provided with NAD and a straight-chain common fatty acid plus ATP and CoA or an acyl-CoA plus CoA. This indicates that the//-oxidation enzyme activities demonstrated in peroxisomes can be linked to form a//-oxidation capacity. Where mitochondria contaminate peroxisomal fractions and if plant mitochondria should possess//-oxidation activity corresponding to that of animal cells (Section V), peroxisomal//-oxidation can be distinguished from mitochondrial fl-oxidation by the KCN-insensitivity of the former system in the presence of NAD, the electron acceptor of the second oxidation step of//-oxidation. The KCN insensitivity of peroxisomal//-oxidation results from the fact that the first oxidation step in peroxisomal//-oxidation is catalyzed by an acyl-CoA oxidase, instead of an acyl-CoA dehydrogenase catalyzing the first oxidation step of//-oxidation in mammalian mitochondria, and being tightly coupled to the electron transport chain of these organelles. As a precaution, experiments on peroxisomal //-oxidation were performed routinely in the presence of KCN or another inhibitor of the mitochondrial electron transport chain. However, there are contradictory reports on inhibition of acyl-CoA oxidase of animal peroxisomes by antimycin A. 59'6° As KCN also inhibits catalase, H202 accumulates in //-oxidation assay mixtures in the presence of KCN. Inhibition of thiolase of rat liver peroxisomes by H20 2 (100 #M, higher than H:O2 concentrations reached in fl-oxidation assay mixtures) has been reported. 61 Rates of//-oxidation were determined frequently as rates of NADH formation and less frequently as rates of acetyl-CoA formation. As NADH formation occurs at the 3-hydroxyacyl-CoA dehydrogenase reaction and, therefore, before the thiolytic cleavage step, NADH formation does not conclusively demonstrate//-oxidation in the strict sense, defined as acetyl-CoA formation from acyl-CoA (fatty acid). This should be borne in mind as regulation of thiolase by intermediates of fl-oxidation is discussed. The results presented in this section relate to experiments performed in hypotonic media and, therefore, on peroxisomes exhibiting low, if any, degree of intactness according to current opinion. (a) Saturated fatty acids. When assaying peroxisomal //-oxidation of straight-chain saturated fatty acids, palmitate or palmitoyl-CoA have been the substrates used preferentially. With respect to these substrates, p-oxidation rates of 0.5-10 nmol NADH formed •sec- ~ • (mg organelle protein)"~ were mainly observed. Few data have been reported on the dependency of p-oxidation rates on the chain length of the straight-chain saturated acyl-CoA used as substrate. Glyoxysomes from cotton cotyledons oxidized butyryl-CoA and palmitoyl-CoA at equal rates (substrate concentrations 200 #M).39 In contrast, the p-oxidation rate of pcroxisomes from castor bean endosperm:s and mung bean hypocotyls62increased up to 4 times when the chain length of the acyl-CoA (concentration 10 #M, below the critical micellar concentration) increased from C4 to C~6. The observed chain length specificity of overall p-oxidation is not cogently predictable from available data on kinetic properties of individual//-oxidation enzymes. Transient accumulation of a short-chain (C4) intermediate was observed in (UJ4C)palmitate oxidation by glyoxysomes from sunflower cotyledons under non-steady state conditions (1-3 #M substrate concentration) (Kleiter and Gerhardt, unpublished data). Besides this intermediate and a Cj6-intermediate (palmitoyl-CoA) accumulating also, only temporarily, no further intermediates were detected by TLC analysis in (U-~4C)palmitate oxidation under non-steady state conditions. The peroxisomal//-oxidation system possesses the ability for complete degradation of long-chain saturated fatty acids to their constituent acetyl units. Using substrate concentrations of 1-3 #M and following product formation until it ceased, it has been demonstrated that 7 nmol NADH. (nmol palmitoyl-CoA)"~ were formed~ and that each nmol (U-~4C)palmitate disappearing from the reaction mixture gave rise to 8 nmol

B. GERHARDT

426

18:3(9 cis,12

cis,15cis)

I

Synthetase

la:3(9

cis, 12 cis, 15 cis}-CoA

12:3(3

ci$,6ci$,9 cls)-CoA

B-Oxldation Isomerase 12:3(2 tran$,6 ci$,9 cl$}-CoA MFP

Thiolaae 10:2(4

ci$,7ci$)-CoA

10:3(2

trans,4 cis,7 cl$)-CoA

Oxidase MFP Thiolaae

/ a:2(2

MFP

~ cla,5 cis)-CoA

~

10:2(3

trans,7ci$}-CoA

I

(D-3-OH)8:I(5

cis)-CoA

10:2(2

I

Dehydratase l 8:2(2

Reductase

trans,5cis)-CoA

8:1(5

Isomerase

trans,7cis)-CoA MFP Thiolase cis)-CoA

Thiolase 6:1(3 ci$)-CoA

Isomerase 6:1(2 trans)-CoA

MFP Thiolase

1 4:0-CoA

B-Oxidation 2:0-CoA FIG. 1. Scheme of peroxisomal linolenate degradaton including the alternative pathways leading

from 2-trans,4-cis,7-cis-deeatrienoyl-CoA to 3-c/s-hexenoyl-CoA.

('4C)acetyl-CoA identified by both chromatographic and enzymatic methods (Kleiter and Gerhardt, unpublished data). (b) Unsaturated fatty acids. Naturally occurring straight-chain unsaturated fatty acids usually have double bond(s) in the c/s configuration which forms a barrier to degradation of these fatty acids by continuous fl-oxidation. The most common unsaturated fatty acids are oleic (18:1, 9 c/s), linoleic (18:2, 9 c/s, 12 cis) and linolenic acid (18:3, 9 c/s, 12 cis, 15 c/s). Figure 1 represents the pathway of linolenate degradation as currently understood. Pathways of oleate and linoleate degradation can be formulated correspondingly to the pathway of linolenate degradation. In the degradation of oleic, linoleic or linolenic acid, both the 9-c/s and 15-cis double bonds of the parent fatty acid are surmounted by the action of A2,A3-enoyl-CoA isomerase (Section IV.A.I.e) at the intermediate level where the double bond has been reached following fl-oxidation. Activity of the isomerase towards 3-c/s-dodecenoyl-CoA, an intermediate in oleate degradation, has been demonstrated with glyoxysomes from cotton cotyledons? 9 The result strongly supports the assumption that the isomerase is also active towards 3-c/s,n-c/s-dodeca-enoyl-CoAs, intermediates in linoleate and linolenate degradation. Due to the 15-c/s double bond of linolenate, 3-c/s-hexenoyl-CoA is formed at the


427

C6-intermediate level in linolenate degradation. Conversion of 3-c/s=hexenoyl-CoA to 2-trans-hexenoyl-CoA has been demonstrated for the isomerase purified from cucumber cotyledons.37 Circumvention of the 12-cis double bond of the parent fatty acid can be achieved by involvement of l>3-hydroxyacyl-CoA dehydratase (Section IV.A.l.f) at the Cs-intermediate level in linoleate and linolenate degradation or by involvement of both 2,4-dienoyl-CoA reductase (Section IV.A.I.g) and isomerase at the C~0-intermediate level (Fig. 1). In the latter case, involvement of isomerase is required as a 3-trans-intermediate is formed by the 2,4-dienoyl-CoA reductase. Activity of 2,4-dienoyl-CoA reductase towards 2-trans,4-transdecadienoyl-CoA has been shown. 37 Activity of D-3-hydroxyacyl-CoA dehydratase has been demonstrated at least towards D-3-hydroxydecanoyl-CoA.38 Using purified enzymes (acyl-CoA oxidase, multifunctional protein, thiolase) in a reconstituted system, conversion of 4-cis-decenoyl-CoA (intermediate in linoleate degradation) to 2-eb-octenoyl-CoA was demonstrated,38 indicating that the plant multifunctional protein is able to convert 2-trans,4-cis-decadienoyl-CoA to 3-hydroxy-4-cis-decenoyl-CoA and that plant thiolase is able to cleave 3-oxo-4-cis-decenoyl-CoA to 2-cis-octenoyl-CoA and acetyl-CoA (cf. Fig. 1). These abilities of plant peroxisomal enzymes are contrary to the abilities of mammalian mitochondrial fl-oxidation enzymes.64'65 Completeness of oleic acid degradation in higher plant peroxisomes was demonstrated using glyoxysomes from sunflower cotyledons. After a lag-phase, continuous formation of (m4C)acetyl-CoA, identified by different methods, from 1 nmol (18-~4C)oleate was observed until the substrate was nearly consumed (Kleiter and Gerhardt, unpublished data). Temporary accumulation of only a few intermediates was observed at (18J4C)oleate degradation under these non-steady state conditions. The intermediates detected were long-chain (oleoyl-CoA), medium-chain and short-chain (C4) intermediates. Complete degradation of linoleate by higher plant peroxisomes is indicated by the following results. (i) (~4C)Citrate formed from oxaloacetate and (14C)acetyl-CoA was the sole end product which accumulated when glyoxysomes from sunflower cotyledons metabolized (U-~4C)linoleate (1 #I~) under non-steady state conditions in the presence of the acetylCoA-removing system (Kleiter and Gerhardt, unpublished data). The amount of (~4C)citrate formed corresponded to that calculated under the assumption that the (U-'4C)linoleate consumed was completely degraded to acetyl-CoA. Transient accumulation of a long-chain (linoleoyl-CoA) and a short-chain (C4-) intermediate was observed as in the cases of degradation of other fatty acids (see above). If acetyl-CoA was not removed from the assay mixture, both acetyl-CoA and a medium-chain intermediate accumulated as end products of linoleate degradation. (ii) (U-~4C)Linoleate degradation by glyoxysomes from cucumber cotyledons under steady-state conditions in the presence of an acetyl-CoA-removing system did not result in intermediate accumulation at the C~2-, C~0- or Cs-level where the barriers to/~-oxidation have to be surmounted. 41 Intermediate accumulation was observed at the C4-1evel. Considering the question of whether linoleate and linolenate are catabolized prevalently via the pathway involving D-3-hydroxyacyl-CoA dehydratase or the pathway involving 2,4-dienoyl-CoA reductase (Fig. 1), there are strong arguments, with respect to higher plant peroxisomes, in favor of the former pathway. (i) Complete degradation of linoleate as well as the rate of linoleate catabolism in glyoxysomes from sunflower cotyledons were uneffected by NADPH (or NADH) required for participation of the 2,4-dienoyl-CoA reductase in linoleate degradation (Kleiter and Gerhardt, unpublished data). (ii) The activity of the D-3-hydroxyacyl-CoA dehydratase in glyoxysomes from cucumber cotyledons was found to be 100 times higher than the 2,4-dienoyl-CoA reductase activity.3s (iii) The low 2,4-dienoyl-CoA reductase activity should lead to intermediate accumulation at, or above, the C~0-1evel; this was not observed.41

428

B. GF.RHARDT

(iv) The hydration of 2-trans,4-cis-decadienoyl-CoA to 3-hydroxy-4-cis-decenoyl-CoA (Fig. 1) which has been considered to be a thermodynamically unfavorable reaction 64 proceeded to an equilibrium with K approximately equal to 1 in glyoxysomes from cucumber cotyledons, 3s allowing entrance into the D-3-hydroxyacyl-CoA dehydratase pathway.

B. Degradation of Ricinoleic Acid Storage triacylglycerols in seeds of certain plants contain appreciable amounts of uncommon fatty acids ~ which are certainly degraded at the mobilization of the reserve lipids during germination. The molecular structure of an uncommon fatty acid may lead to interruption of degradation of the fatty acid by repetitive passages through the E-oxidation reaction sequence. So far, degradation of ricinoleic acid (D-12-hydroxy-9-cisoctadecenoic acid) has only been studied in detail, u' 45 Ricinoleic acid comprises more than 80 % of the fatty acid moieties of the storage triacylglycerols in the castor bean endosperm. Localization of ricinoleate degradation in peroxisomes has been demonstrated and it is established that peroxisomes/cell-free systems from tissues which do not contain ricinoleic acid possess also the capacity to degrade this uncommon fatty acid. u' 4s Ricinoleic acid is activated in a reaction indicating involvement of acyl-CoA synthetase. 42'44 Rates of activation were comparable to those of palmitic acid activation. Up to the Cl0-intermediate level, the intermediates formed by successive removal of C2-units from ricinoleoyl-CoA have been detected by GLC analysis and the C2-units released were identified as acetyl-CoA. 4s As ricinoleic acid is a hydroxy analog of oleic acid, a 3-c/s-intermediate forming a barrier to further E-oxidation results at the CI2-intermediate level. The original conclusion 4s that the intermediate D-6-hydroxy-3-cis-dodecenoyl-CoA at ricinoleate degradation is converted into D-6-hydroxy-2-trans-dodecenoyl-CoA by an isomerase is strongly supported today since an isomerase acting also towards mediumchain enoyl-CoAs has been purified from cucumber cotyledons. 37 Based on experimental evidence, Hutton and StumpP 5 could not conclusively resolve the pathway of ricinoleate degradation at the Cs-intermediate level where the hydroxyl group of ricinoleic acid forms a barrier to further degradation of ricinoleic acid by/~-oxidation. At the Cs-intermediate level, 2-hydroxyoctanoate and 2-oxo-octanoate were detected (for methodological reasons, the studies did not distinguish between intermediates esterified or not esterified to CoASH). It was also demonstrated that 2-oxo-octanoate was metabolized beyond the Cs-level and that C-3 of this compound became C-2 of acetate. Based on these data, it was tentatively suggested that an a-oxidation step was involved at the Csintermediate level in order to surmount the/~-oxidation barrier caused by the hydroxyl group. The a-oxidation would yield CO2 and heptanoate which can be degraded further by E-oxidation. Involvement of g-oxidation (in the sense that this process is generally understood; Section VI) in ricinoleate degradation at the Cs-intermediate level would imply: (i) the acyl-CoA track of E-oxidation has to be left at the C8-intermediate level since ~-oxidation acts on substrates (fatty acids) not esterified to CoASH; (ii) the heptanoate formed by ~-oxidation has to be newly activated by acyl-CoA synthetase, consuming ATP, in order to allow the return to the acyl-CoA track of -oxidation; Off) a-oxidation considered to be located at the endoplasmic reticulum has to be localized (also) in peroxisomes since the organelles completely degrade ricinoleate to acetyl-CoA and propionyl-CoA (see below). In addition, 2-hydroxy acid is currently considered to be a deadend by-product of ~-oxidation and not to be an intermediate of this process. 67 The unresolved problem of ricinoleate degradation at the Cs-intermediate level has been reinvestigated in recent studies. 44 Using HPLC analyses differentiating between acyl-CoA thioester intermediates and intermediates not esterified to CoASH, two intermediates not esterified to CoASH were detected at ricinoleate degradation by peroxisomes. These


429

intermediates were 2-hydroxyoctanoate and 2-oxo-octanoate. Heptanoate was not detected (although the resolution limits of the HPLC procedure allowed detection of heptanoate in a mixture composed of hexanoate, heptanoate, octanoate, 2-hydroxyoctanoate, and 2-oxo-octanoate). 2-Hydroxyoctanoate was oxidized by peroxisomes to 2-oxo-octanoate in a reaction generating H202 and showing a stoichiometry of H202 and 2-oxo-octanoate formation of 1:1. The characteristics of this oxidation reaction indicate involvement of a 2-hydroxy acid oxidase. The higher plant/peroxisomal 2-hydroxy acid oxidases, known so far, act preferentially on L-2-hydroxy acids while the 2-hydroxyoctanoate formed as intermediate at ricinoleate degradation should have the D-configuration. However, both isomers of racemic 2-hydroxyoctanoate used as substrate were oxidized by the peroxisomes. 2-Oxo-octanoate was metabolized to heptanoyl-CoA (and degraded further to propionyl-CoA and acetyl-CoA). The formation of heptanoyl-CoA from 2-oxo-octanoate could result from either an acyl-CoA synthetase reaction acting on heptanoate generated somehow from 2oxo-octanoate, or an oxidative decarboxylation of 2-oxo-octanoate releasing directly heptanoyl-CoA. The ability of peroxisomes to perform oxidative decarboxylations has been demonstrated. 6s There are several arguments in favor of an oxidative decarboxylation of the 2-oxooctanoate generated as intermediate at the Cs-lcvel of ricinoleate degradationO: (i) heptanoyl-CoA was formed in an ATP-independent, NAD-dependent reaction; (ii) heptanoate was not detected as intermediate when 2-oxo-octanoate was metabolized to heptanoyl-CoA (the metabolism of which was prevented) although the concomitant N A D H formation was 2 times higher than the acyl-CoA synthetase activity assayed with heptanoate as substrate; (iii) ricinoleate or 2-oxo-octanoate metabolism was not inhibited by imidazole, an inhibitor of ~-oxidation, but 2-oxo-octanoate accumulated as end product of ricinoleate degradation in the presence of arsenite, an inhibitor of oxidative decarboxylation. The pathway leading from the Cl0-intermediate level to heptanoyl-CoA at ricinoleate degradation is shown in Fig. 2 as currently proposed. *~ The alternative reaction sequence [Ricinoleoyl-CoA]

B-Oxidation

I D-4-Hydroxy-10:0-CoA

B-Oxidation D-2-Hydroxy-8:o-CoA

Hydrolase

4-Oxo-10:0-CoA D-2-Hydroxy-8:0

2-Hydroxyacid oxidase

[ 2-Oxo-8:0

•

Oxidative [ decarboxylation 7:0-CoA

B-Oxidation

I 3:0-CoA

FIG.2. Proposed pathways leading from D-4-hydroxydecanoyl-CoA to heptanoyl-CoA at peroxisomal ricinoleoyl-CoA degradation (adapted from Refs 44 and 45).

B. GERHARDT

430

leading from D-4-hydroxydecanoyl-CoA via 4-oxodecanoyl-CoA to 2-oxo-octanoate has been included since 4-oxo-octanoate (4-oxo-octanoyl-CoA) has been reported to be also an intermediate at ricinoleate degradation. 45 Propionyl-CoA is metabolized to acetyl-CoA in peroxisomes (Section IV.C) and is an intermediate accumulating only transiently at ricinoleate catabolism. 44 Under non-steady state conditions, acetyl-CoA was detected as sole product of ricinoleate degradation when the concomitant NADH formation had ceased. The amount of 9 nmol N A D H formed corresponded to that expected for complete degradation of 1 nmol ricinoleate to acetyl-CoA by the proposed pathway.

C. Degradation of Propionyl-CoA Catabolism of odd-numbered, straight-chain fatty acids by fl-oxidation results in the formation of propionyl-CoA. But, such fatty acids are rarely found in plant tissues as constituents of storage triacylglycerols or membrane lipids. However, propionyl-CoA is generated in certain catabolic processes such as degradation of branched-chain 2-oxo acids or uncommon fatty acids. A pathway of propionyl-CoA degradation in plants has been proposed by Stumpf and coworkers69. 70 and was called modified fl-oxidation. The proposal (Fig. 3) was primarily based on the following results: (i) CO2 was not required for degradation of propionate in cell-free systems from plants in contrast to its requirement for propionate degradation in mammalian cells carboxylating propionyl-CoA to methylmalonyl-CoA which is subsequently rearranged to form succinylCoA;

CH3-CH-COSCOA I R

1

Oxidase

CH2=C-COSCoA I R

l

MFP

CH2-CH-COSCoA I

I

OH

R

1

Hydrolase

CH2-CH-COOI

I

OH

R

1

Dehydrogenase

CH-CH-COOII

I

O R

1

CoASCO-CH2 + CO2 I

R FIo. 3. Pathwayof modified/~-oxidation(adapted from Refs70, 72 and 73). R =--H, propionylCoA; R =----CH3, isobutyryl-CoA.MFP--multifunctionalprotein.


431

(ii) C-I of propionate was metabolized to CO2 and C-2 and C-3 of propionate became C-2 and C-l, respectively, of acetyl-CoA; (iii) 3-hydroxypropionate was demonstrated as an intermediate of propionate degradation. Apart from 3-hydroxypropionate, none of the intermediates of the proposed modified fl-oxidation had (directly) been demonstrated. Degradation of propionate via 3-hydroxypropionate to acetate has also been reported recently.71 The pathway of propionate degradation has been reinvestigated very recently, suggesting its localization in peroxisomes where propionyl-CoA is formed at ricinoleate degradation (Section IV.B) and the catabolism of branched-chain 2-oxo acids (Section IV.D). Peroxisomes from mung bean hypocotyls degraded propionyl-CoA to acetyl-CoA.72 Following propionyl-CoA degradation in kinetic experiments, all intermediates (acrylylCoA, 3-hydroxypropionyl-CoA, 3-hydroxypropionate, and malonic semialdehyde) of the pathway of modified fl-oxidation (Fig. 3) were detected using HPLC analyses: 2'73 Malonyl-CoA, a possible intermediate of modified fl-oxidation, was not demonstrated unequivocally and succinyl-CoA, product of propionyl-CoA metabolism in mammalian mitochondria, was not detected. Propionate was activated to propionyl-CoA by peroxisomes from mung bean hypocotyls in a reaction indicating involvement of acyl-CoA synthetase. The rate of propionate activation amounted to approximately one-third of palmitate activation. 72 The rate of propionyl-CoA oxidation to acrylyl-CoA amounted to approximately two-thirds of the rate at which palmitoyl-CoA was oxidized to 2,3-dehydropalmitoyl-CoA. As the oxidation of propionyl-CoA to acrylyl-CoA resulted in concomitant H202 formation, the oxidation appears to be catalyzed by acyl-CoA oxidase. The hydration of acrylyl-CoA to 3-hydroxypropional-CoA may be catalyzed by the multifunctional protein. According to kinetic characteristics of the enzyme currently available (Section IV.A.I.c) the enoyl-CoA hydratase activity appears to be at least as active towards short-chain as towards long-chain enoyl-CoAs. The enzymes involved in the reaction sequence leading from 3-hydroxypropionyl-CoA to acetyl-CoA plus CO2 have not yet been studied. The dehydrogenase catalyzing the oxidation of 3-hydroxypropionate to malonic semialdehyde uses NAD as electron acceptor. In the absence ofNAD, 3-hydroxypropionate accumulated as end product of propionyl-CoA degradation. 73 Correspondingly, malonic semialdehyde accumulated when CoASH was omitted from the reaction mixture. Degradation of propionate/propionyl-CoA in higher plants by modified fl-oxidation proposed originally by Stumpf and coworkers69"7o has been confirmed nowadays and localization of modified p-oxidation in peroxisomes has been demonstrated72"73 However, there are a few older reports suggesting that propionate may additionally be metabolized, in potato tubers, by a pathway similar to that occurring in mammalian mitochondria. The suggestion is based on results of labelling experiments74 and the detection of methylmalonyl-CoA mutase activity. 75 If the additional pathway of propionate degradation occurs in potato tubers (and other plant organs/tissues), its subcellular localization is unknown at present.

D. Degradation of Branched-Chain 2-Oxo Acids Branched-chain 2-oxo acids are intermediates at the catabolism of leucine, isoleucine, and valine and are generated when these amino acids undergo aminotransferase reaction. As catabolism of the branched-chain amino acids in plants has received very little attention, pathways of branched-chain 2-oxo acids (amino acids) in plants were thought to correspond to those established in mammalian cells. It was only very recently that degradation of branched-chain 2-oxo acids in plants has been studied in some detail, s' 68. 73,76.77 Activation and subsequent degradation of branched-chain 2-oxo acids were demonstrated to be localized in peroxisomes from mung bean hypocotyls and, so far, reported data on branched-chain 2-oxo acid activation and degradation relate to studies performed with these peroxisomes. JPLR 31/4----O

432

B.G.mU~dtDT

1. Activation by Oxidative Decarboxylation The branched-chain 2-oxo acids 2-oxoisocaproic, 2-oxo-3-methylvaleric and 2-oxoisovaleric acid are generated from leucine, isoleucine and valine, respectively, by aminotransferase reaction. The branched-chain 2-oxo acids are activated for degradation by oxidative decarboxylation as demonstrated by cofactor requirements and analysis of the products of the reaction acting on the branched-chain 2-oxo acids in peroxisomes.6s Peroxisomal metabolism of the branched-chain 2-oxo acids required CoASH and NAD; it resulted in formation of NADH, 14CO 2 release from C-1 labelled branched-chain 2-oxo acid, and formation of an acyl-CoA containing one carbon atom less than the branched-chain 2-oxo fatty acid used as substrate. The products of the reaction were formed in a 1:1:1 stoichiometry. Rates of oxidative decarboxylation of 50-100 pkat.(mg peroxisomai protein)"~were observed. The branched-chain 2-oxo acid dehydrogenase complex supposed to catalyze the observed peroxisomal reaction has not yet been characterized or purified from a plant source.

2. Pathways of Degradation The proposed pathways of branched-chain 2-oxo acid degradation which are outlined in the following are primarily based on identification of intermediates by HPLC analyses using reference standards and kinetic experiments. The pathways will be considered only up to the formation of propionyl-CoA, an intermediate in catabolism of the branchedchain 2-oxo acids studied. Propionyl-CoA was, however, traceably catabolized further to acetyl-CoA.76 So far, individual reactions of the proposed pathways have been analyzed only in a few cases. It is unknown at present whether fl-oxidation reactions involved in the catabolism of branched-chain acyl-CoA thioesters are catalyzed by the proteins acting on straight-chain acyl-CoA thioesters at fl-oxidation. As certain reference standards, for HPLC analyses, of branched-chain acyl-CoA thioesters were synthesized using purified fl-oxidation enzymes from animal sources, the fl-oxidation enzymes acting on straightchain acyl-CoA thioesters show at least activity towards branched-chain acyl-CoA thioesters. Oxidation of the branched-chain acyl-CoAs to the corresponding 2-enoyl-CoAs resulted in concomitant H~O2 formation, indicating that the reaction was catalyzed by acyl-CoA oxidase. 68 (a) Degradation of 2-oxo-3-methylvalerate. Following oxidative decarboxylation of 2-oxo-3-methylvalerate formed by transamination of isoleucine the acyl-CoA generated is degraded to propionyl-CoA plus acetyl-CoA by one complete passage through the ]/-oxidation reaction sequence. All intermediates of this fl-oxidation have been demonstrated. 76 (b) Degradation of 2-oxoisovalerate. Oxidative decarboxylation of 2-oxoisovalerate generated by transamination of valine results in the formation of isobutyryl-CoA (2-methylpropionyl-CoA) which is degraded to propionyl-CoA by modified fl-oxidation (Fig. 3), the pathway of propionyl-CoA degradation. The acyl-CoA thioester intermediates and the nonesterified intermediates occurring at isobutyryl-CoA degradation by modified fl-oxidation have been demonstrated.73,76 However, neither conversion of C-1 of isobutyryl-CoA to CO2 nor CO 2 evolution were demonstrated at isobutyryl-CoA degradation. As in the case of propionyl-CoA degradation, the intermediates not esterified to CoASH, i.e. 3-hydroxyisobutyrate and methylmalonate semialdehyde, accumulated at isovalerylCoA degradation when NAD and CoASH, respectively, were omitted from the reaction mixture. 73

(c) Degradation of 2-oxoisocaproate. Figure 4 shows the peroxisomal pathway of 2-oxoisocaproate degradation as proposed according to current experimental data.

Fatty acid degradafionin ~ants

433

2-Oxoisocaproate Oxidative decarboxylation Oxidase

3-Methyl-4:l-CoA (3-Methylcrotonyl-CoA)

1

3-Methyl-4:l (Senecioate)

1

2-Hydroxy-3-methyl-4:0 (2-Hydroxyisovalerate) 2-Hydroxy acid oxidase

1

2-Oxo-3-methyl-4:0 (2-Oxoisovalerate) Oxidative decarboxylation

1

2-Methyl-3:0-CoA (Isobutyryl-CoA) Modified B-Oxidation

1

Propionyl-CoA FIG. 4. Proposed pathway for peroxisomal degradation of 2-oxoisocaproate to propionyl-CoA (adapted from Refs 68, 76 and 77).

Following oxidative decarboxylation of 2-oxoisocaproate generated by transamination from leucine the isovaleryl-CoA formed is subsequently oxidized to 3-methylcrotonyl-CoA by acyl-CoA oxidase. ~,76 However, the methyl group at the C-3 position forms a barrier to further degradation by//-oxidation, in contrast to a methyl group positioned at the C-2 of the fatty acid carbon chain as in the case of the intermediates methacrylyl-CoA (2-methylpropenoyl-CoA) and tiglyl-CoA (2-methyl-2-butenoyl-CoA) at degradation of 2-oxo-isovalerate (Fig. 3) and 2-oxo-3-methylvalerate, respectively. Hydration of 3-methylcrotonyl-CoA by the multifunctional protein leads to 3-hydroxy-3-methylbutyryl-CoA, a deadend product with respect to basic //-oxidation. In mammalian mitochondria, the barrier formed by the methyl group of 3-methylcrotonyl-CoA is surmounted by metabolism of 3-methylcrotonyl-CoA to HMG-CoA, involving a carboxylation reaction. HMG-CoA was not detected when 2-oxoisocaproate or isovaleryl-CoA were degraded by peroxisomes although it was detected in cell free extracts. 76 Peroxisomes metabolized 2-oxoisocaproate/isovaleryl-CoA to isobutyryl-CoA and acyl-CoA thioester intermediates were not detected, except 3-methylcrotonyl-CoA. 76 Detected were senecioate, 2-hydroxyisovalerate and 2-oxoisovalerate. Sequential conversion of these intermediates and their degradation to isobutyryl-CoA were demonstrated, 77 suggesting that a reaction sequence leads from 3-methylcrotonyl-CoA to isobutyryl-CoA at 2-oxoisocaproate degradation as shown in Fig. 4. Oxidation of 2°hydroxyisovalerate to 2-oxoisovalerate occurred in a H202-generating reaction and the conversion of 2-oxoisovalerate to isobutyryl-CoA

434

B. G ~ t . ~ D ' r

required NAD and CoASH (Gerbling and Gerhardt, unpublished data), indicating involvement of 2-hydroxy acid oxidase and oxidative decarboxylation, respectively. While the reaction(s) introducing the hydroxyl group at C-2 of senecioate are still unknown, the subsequent reaction sequence leading from 2-hydroxyisovalerate to isobutyryl-CoA appears to correspond to that involved in ricinoleate degradation at the Cs-intermediate level (Section IV.B). Isobutyryl-CoA is degraded to propionyl-CoA by modified//-oxidation (Section IV.D.2.b) The proposed pathway of 2-oxoisocaproate degradation in higher plant peroxisomes differs considerably from the pathway of 2-oxoisocaproate degradation in mammalian mitochondria. In contrast, the pathway of 2-oxoisovalerate degradation in higher plant peroxisomes largely corresponds to, and that of 2-oxo-3-methylvalerate degradation is in accordance with, the corresponding pathway in mammalian mitochondria. E. Particularities of the Peroxisomal Fatty Acid Degrading System Fatty acid degradation in higher plant peroxisomes is characterized by certain particularities if compared to the classic fatty acid degrading (B-oxidation) system located in mammalian mitochondria but, so far, not demonstrated unequivocally in mitochondria of higher plants (Section V), or if compared to peroxisomal B-oxidation systems of other organisms. The particularities are only summarized here without presenting experimental evidence which has been treated in the foregoing sections. The particularities outlined under (a) are general characteristics of the peroxisomal B-oxidation system, these presented under (b), (c) and (d) are specific to higher plants in some respect. Whether the other particularities (e-g) are compartment-specific or specific to higher plants cannot be decided at present as these particularities belong to pathways which have not yet been studied in peroxisomes from heterotrophic organisms. (a) The first oxidation step of the basic B-oxidation reaction sequence is catalyzed in peroxisomes by an acyl-CoA oxidase instead of an acyl-CoA dehydrogenase involved in mitochondrial B-oxidation systems. The two subsequent reactions, the hydration step and the second oxidation step, are catalyzed by a multifunctional protein while each of these reactions is catalyzed by an individual protein in mitochondrial systems. Both acyl-CoA oxidase and multifunctional protein are characteristic of the peroxisomal B-oxidation system in general as they are also involved in the peroxisomal B-oxidation in animal cells and yeasts. 9'49'78'79 However, the multifunctional proteins of higher plant and yeast peroxisomes differ from the multifunctional protein of mammalian peroxisomess° inasmuch as the former exhibit "epimerase" activity instead of isomerase activity in addition to the 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activity. (b) According to their enzymic constituents, higher plant peroxisomes are capable of degrading unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms by both the 2,4-dienoyl-CoA reductase pathway and the D-3-hydroxyacylCoA dehydratase pathway (Fig. 1.). It appears, however, that the D-3-hydroxyacyl-CoA dehydratase pathway is predominantly, if not exclusively, active. Mammalian and yeast peroxisomes have also the capability to perform both pathways according to their enzymic constituents. But, the 2,4-dienoyl-CoA reductase pathway appears to be predominant in these peroxisomes. 64"st Mammalian mitochondria lack D-3-hydroxyacyl-CoA dehydratase and, therefore, can perform only the 2,4-dienoyl-CoA reductase pathway. 9"~ (c) Higher plant peroxisomes are capable of degrading completely long-chain fatty acids to their constituent acetyl-units while the peroxisomal B-oxidation system of animal cells appears to be rather inactive towards short-chain fatty acids and to function as a chain-shortening system. ~°,s2 Since yeast cells have been reported to possess exclusively the peroxisomal B-oxidation system,83 complete degradation of long-chain fatty acids by this system should occur also in these organisms. (d) The peroxisomal B-oxidation system of higher plant cells is carnitine-independent (Section IV.H). Mammalian peroxisomes possess carnitine acyltransferases; however, their functions are not clear at present. '°


435

(e) Higher plant peroxisomes are apparently unable to carry out both biotin-dependent carboxylations and coenzyme Bl:-dependent mutase reactions. These reaction types are involved in propionyl-CoA and 2-oxoisoeaproate (isovaleryl-CoA) degradation and propionyl-CoA and 2-oxoisovalerate (isobutyryl-CoA) degradation, respectively, in mammalian mitochondria. These compounds are catabolized in higher plant peroxisomes by biotin- and coenzyme Bn-independent pathways. (f) The peroxisomal system of higher plant cells degrades propionyl-CoA and isobutyrylCoA by the pathway of so-called modified ]/-oxidation unknown in mammalian tissues (although degradation of isobutyryl-CoA proceeds in mammalian mitoehondria by a pathway resembling partly modified//-oxidation). (g) 2-Hydroxy acids, intermediates formed at degradation of certain fatty acids, are oxidized at the g-carbon atom by a peroxisomal 2-hydroxy acid oxidase and the 2-oxo acids formed undergo oxidative decarboxylation allowing return to the acyl-CoA track of ]/-oxidation. The oxidation mechanism acting on the a-carbon atom is different from that known as a-oxidation.

F. Fate of Acetyl-CoA With respect to carbon, peroxisomal degradation of physiologically relevant fatty acids in higher plant cells leads to acetyl-CoA as sole end product, apart from CO2 evolved at oxidative decarboxylations. The acetyl-CoA is metabolized within the peroxisomes. Its fate depends on the enzymic composition of the peroxisomes which in turn depends on the physiological and/or developmental status of the cell) Enzymes known to be active towards acetyl-CoA in higher plant peroxisomes lacking acetyl-CoA hydrolase activity are citrate synthase and malate synthase. According to current knowledge, an isoform of citrate synthase appears to be a regular constituent of higher plant peroxisomes 8"25'26's5'86 while malate synthase is restricted to glyoxysomes performing the glyoxylate cycle/ pathway. Metabolism of acetyl-CoA via the glyoxylate cycle/pathway in glyoxysomes (Section III) 12's7 results in the net production of C4-dicarboxylic acid released from the organdies and directed into gluconeogenesis. Whether the glyoxylate cycle1: or the glyoxylate pathway, 87 the two ways in which the reactions involved in acetyl-CoA metabolism in glyoxysomes have been arranged diagrammatically, mirror the reality has yet to be established. A pathway for acetyl-CoA metabolism in nonglyoxysomal peroxisomes (Section III) has been proposed very recently,s's6'as According to the proposal, acetyl-CoA is metabolized by citrate synthase to citrate which is converted to isocitrate and subsequently decarboxylated to 2-oxoglutarate by NADP-dependent isocitrate dehydrogenase. The oxaloacetate required for citrate synthesis is generated by an aminotransferase acting on the 2-oxogiutarate formed within the organelles and aspartate imported into the organelles in exchange for the glutamate formed from 2-oxoglutarate in the aminotransferase reaction. Thus, the carbon of acetyl-CoA becomes part of exported glutamate in nonglyoxysomal peroxisomes. Peroxisomal isoforms of the enzymes required for the proposed pathway were demonstrated, in purified potato tuber and other nonglyoxysomal peroxisomes, on the basis of their specific elution patterns from hydroxylapatite columns and their specific properties; an isoform of aconitase was not identified because of the high instability of aconitase activity. The aconitase activity of the peroxisomal fractions differed, however, in its properties from the mitochondrial aconitase activity. Feeding of isocitrate, NADP, aspartate and acetyl-CoA to potato tuber peroxisomes resulted in CoASH liberation, citrate and glutamate formation in a 1:1:1 stoichiometry (aconitase inactivated). Present conceptions of acetyl-CoA metabolism in both glyoxysomes and nonglyoxysomal peroxisomes involve aconitase. However, occurrence of this enzyme in peroxisomes has been questioned very recently89 since mitochondrial aconitase was found to be inactivated by (100 #M) H20~ formed in peroxisomes, for example, at the acyl-CoA oxidase reaction and generally thought to be decomposed by the peroxisomal maker catalase

436

B. GERI-IARDT

amounting to up to 20% of peroxisomal protein. If aconitase activity detected in peroxisomal fractions is due to contamination by mitochondria and/or cytosolic aconitase, i.e. if peroxisomes lack aconitase, the carbon of acetyl-CoA generated at fatty acid degradation is exported from the peroxisomes following citrate formation. Metabolic consequences resulting from such a situation are not a topic of discussion here. G. Energetic Aspects Fatty acid degradation proceeds with concomitant energy release. Energy conservation in form of ATP generation is unknown in peroxisomes. The energy released at the oxidase reaction (AH = - 84 kJ mol - ~) and the catalase step (AH = - 26 kJ mol- I) is lost as heat. NAD(P)H formed within peroxisomes does not appear to leave the organelles for subsequent extraperoxisomal oxidation. Latency of NADH-dependent enzyme activities in higher plant peroxisomes has been observed 87,9° indicating that restrictions to direct permeation of N A D H across the peroxisomal membrane exist since metabolites of low molecular weight appear to cross the membrane without restrictions in vitro, s7 However, established knowledge is lacking regarding permeability properties of and transport systems in the membrane of higher plant peroxisomes. NAD(P)H formed within higher plant peroxisomes is thought to be reoxidized within the organdies in order to sustain fatty acid degradation (and other NAD(P)H generating processes in peroxisomes). A NAD level of 0.2-0.6 nmol NAD. (mg organdie protein) "~ has been determined in castor bean endosperm glyoxysomes isolated by aqueous methods, s7'91 Two mechanisms, a malate-aspartate shuttle transferring reducing equivalents across the peroxisomal membrane (and into mitochondria) 2s's7 and a membrane located electron transport system63'92'93 have been proposed for reoxidation of N A D H generated in glyoxysomes. Both concepts are supported by experimental evidence obtained with castor bean endosperm glyoxysomes so far. The two routes of intraperoxisomal N A D H oxidation may operate in parallel or alternately. The membrane located electron transport system includes a flavoprotein NADH reductase and cytochrome bs. The orientation of the components within the peroxisome membrane allows electron uptake from the organelle matrix and electron discharge into the cytosol. 94 Thus, the electron transport system appears to function in transferring electrons across the glyoxysomal membrane. The cytosolic electron acceptor is as yet unknown. Whether the electron transfer across the glyoxysomal membrane leads to energy conservation depends on its nature. Oxidation of palmitoyl-CoA has been coupled via the membrane located electron transfer system to the reduction of artificial electron acceptors. 63 A malate-aspartate shuttle operating between glyoxysomes and mitochondria can link glyoxysomal fatty acid degradation to mitochondrial ATP formation. With respect to nonglyoxysomal peroxisomes in which N A D H formation resulting from fatty acid degradation is certainly low, there is evidence that leaf peroxisomes are provided with N A D H by a malate-oxaloacetate/aspartate shuttle at photorespiration. 95'9~ As the NADH-consuming part (glycerate pathway) of photorespiration is reversible97 the shuttle should also function in the reverse direction in the dark. However, peroxisomal NADH oxidation by a malate-aspartate shuttle has to be generalized with caution at present, since certain nonglyoxysomal peroxisomes have been reported to lack malate dehydrogenase. H. Transport of Fatty Acids Fatty acids originate by the activity of lipases from storage triacylglycerols accumulated in oil bodies, or at membrane lipid turnover/degradation. So far, it is virtually unknown as to how fatty acids reach the peroxisomes from their point of origin. Mediation of transfer by membrane contact between oil bodies and peroxisomes or involvement of lipid transfer/fatty acid binding proteins are at best speculative, and the latter is rather unlikely. 9s


437

Prior to activation, fatty acids have to cross the peroxisome membrane since acyl-CoA synthetase of higher plant peroxisomes appears to be located at the inner face of the organdie membrane (Section IV.A. 1.a). How fatty acids cross the peroxisome membrane is unknown. A carnitine transfer system does not appear to be involved. Acylcarnitines are not oxidized by higher plant peroxisomes 28.39.62and carnitine acyltranferase activities have not been detected in the organelles. 62'99 I. Regulation

So far, information on regulation, at different levels of fatty acid degradation in higher plant peroxisomes exists only sporadically and is also of indirect evidence in many cases. According to the data presented in Table 2, thiolase seems mainly to be the rate-limiting enzyme of the/~-oxidation reaction sequence. However, one has to bear in mind that the individual enzymes involved in fatty acid degradation were assayed using substrates of different chain length: C~6-substrates in the case of acyl-CoA synthetase and acyl-CoA oxidase; C4-substrates in the case of multifunctional protein and thiolase. In addition, 3-hydroxyacyl-CoA dehydrogenase activity was generally assayed in the reverse direction. Nevertheless, in the few cases where data on acyl-CoA oxidase activity towards butyrylCoA have been reported (Section IV.A.I.b), these activities, when lower than those towards palmitoyl-CoA, were not lower than thiolase activities. Available data on the rate of sucrose formation from fat in the castor bean endosperm allow the estimation of the fl-oxidation enzyme activities required in vivo to sustain this rate. These activities may be compared with the activities determined in vitro. At the peak of fat breakdown, 33 nmol acetyl-CoA'sec-l'(mg organelle protein) -~ have to be generated in vivo to sustain the rate of hexose formation. 25 Activation of ricinoleate by acyl-CoA synthetase in vitro amounts to approximately 40% of the activity required in vivo (4 nmol ricinoleate activated.sec -~ .(mg organdie protein)-~). In contrast, acyl-CoA oxidase which has to metabolize 33 nmol substrate, sec-1. (mg organelle protein)- ~in vivo showed approximately one-tenth of this activity towards ricinoleoyl-CoA in vitro (Section IV.B). Rates of overall fl-oxidation determined in vitro with ricinoleate or ricinoleoyl-CoA as substrate were even less. Transient accumulation of a few intermediates was observed when peroxisomes from sunflower cotyledons degraded (U-14C)palmitate, (18-~4C)oleate or (UJ4C)linoleate under non-steady state conditions (Section IV.A.2). Transient accumulation of the activated substrates was to be expected since the acyl-CoA oxidase activity towards acyl-CoAs of decreasing chain-length was on the average hardly 7 times as large as the activity of acyl-CoA synthetase (Table 2). Substrate channelling from one passage through the fl-oxidation reaction sequence to the next passage could additionally be involved in the transient accumulation of the activated substrates. The transient accumulation observed at the C4-intermediate level under non-steady state conditions of fatty acid degradation as well as the C4-intermediate accumulation under steady state conditions of fatty acid degradation by glyoxysomes from cucumber cotyledons*1 indicate that degradation of long-chain fatty acids generally appears to slow down at the C4-intermediate level. In contrast to complete degradation of palmitate and oleate, complete degradation of linoleate required removal of acetyl-CoA from the assay mixture, otherwise an intermediate of medium chain length accumulated. The point of regulation has yet to be elucidated but may be located at the Ci0-/Cs-intermediate level where the fl-oxidation barrier caused by the 12-cis double bond of linoleate has to be surmounted. During germination of oilseeds, the fl-oxidation enzyme activities rise and fall in the lipid-mobilizing tissue of the seeds. 39'1°1'1°2 The processes which underlie these temporal changes in fl-oxidation enzyme activities have been studied only sporadically. However, an increasing amount of data demonstrates that peroxisomal enzyme activities rise, during germination, due to increased transcriptional activity although processes operating at other levels can also be involved, m°3-1°5It seems likely then that the rise in fl-oxidation enzyme activities during germination (and that reported for senescing leaves23) is primarily due

438

B. GERHARDT

to increased expression of the genes encoding fl-oxidation enzyme proteins. As all peroxisomal proteins studied under the aspect of biosynthesis have been shown to be synthesized on free polyribosomes and to be inserted post-translationally into the organdie, fl-oxidation enzyme proteins are certainly synthesized in the cytosol. Detection of pulse-labeled multifunctional protein in the cytosol prior to the appearance of the protein in glyoxysomes has been reported.I°6 Loss in fl-oxidation enzyme activities at later stages of oilseed (cucumber) germination is due to both a decrease in the amount of encoding RNAs and protein degradation/turnover) °7 So far, experimental evidence is virtually lacking for both the nature of the stimuli causing changes in transcriptional activity concerning fl-oxidation enzyme proteins, and signal transduction. Light appears to be involved somehow in down-regulation of fl-oxidation enzyme activities at later stages of oilseed germination. V. MITOCHONDRIAL fl-OXIDATION IN HIGHER PLANTS After the discovery of glyoxysomes, it was demonstrated that these organelles are the subcellular site of fatty acid degradation (fl-oxidation) in lipid-mobilizing tissues of oilseeds. However, it was still thought that mitochondria are the site of fl-oxidation in the majority of higher plant tissues4 although there was hardly any experimental evidence for that. Today, it is well established that higher plant peroxisomes, regular constituents of higher plant cells, are generally able to degrade fatty acids. Thus, does a mitochondrial fl-oxidation system exist in higher plant cells in addition to the peroxisomal fl-oxidation system or do higher plant mitochondria lack fl-oxidation activity? Since the demonstration of peroxisomal fl-oxidation in higher plant cells, it has repeatedly been reported that mitochondria from pea cotyledons (and also from some other tissues) are able to perform fl-oxidation. The studies started from different approaches. However, the ability of pea cotyledon (higher plant) mitochondria to perform fl-oxidation has to be questioned on the basis of certain data. In the following, arguments in favor of mitochondrial//-oxidation in pea cotyledons will be considered alongside counter-arguments. Peroxisomal fl-oxidation has been demonstrated in pea cotyledons. 31'~°s Dual location of ]/-oxidation, in mitochondria and peroxisomes, is well established for mammalian cells and the two fl-oxidation systems differ in their properties and functions. 9

A. Carnitine Acyltransferase L-Carnitine has been demonstrated in plant tissues ~°9't1° and carnitine acyltransferase activity has been detected in higher plant mitochondria, using long-, medium- and short-chain acyl-CoAs as substrates. 99"1°8'm-113The carnitine acyltransferase activities of pea cotyledon mitochondria which are detectable with long- or short-chain acyl-CoAs have optionally been ascribed to two different enzymes with different functions, t°s'N2'113 The enzyme thought to react with long-chain acyl-CoAs is thought to be involved in mitochondrial fl-oxidation) °8'113 The sole indication of two carnitine long-chain and short-chain acyltransferases in pea cotyledon mitochondria rests on results of latency experiments performed, however, not in parallel. The carnitine acyltransferase of mitoplasts ("mitochondria" without outer membrane) showed nearly complete latency towards acetyl-CoA 112 but only partial latency towards palmitoyl-CoA. I°s In the later case, intactness of the mitoplasts, i.e. of the inner mitochondrial membrane was not demonstrated.rOB The presumed carnitine acetyltransferase is thought to be located only inside the inner mitochondrial membrane; the presumed carnitine long-chain acyltransferase is thought to be located intramitochondrially as well as at the outer face of the inner mitochondrial membrane (corresponding to the situation in mammalian mitochondria). Studies on the mitochondrial carnitine acyltransferase activity of mung bean hypocotyls suggested that the activity may be due to a single protein resembling a short-chain rather than a long-chain carnitine acyltransferase. 99'114 For example, using different acetyl-CoA and palmitoyl-CoA concentrations in competition experiments, the activity observed in the


439

presence of both substrates at saturating concentrations reached but did not exceed the activity observed at saturating acetyl-CoA concentration although the acylcarnitines of both substrates were formed. 99 An answer to the question of whether plant mitochondfia possess different carnitine acyltransferases has to await purification of the enzyme(s). It can only then be decided whether the demonstrated carnitine long-chain acyltransferase activity is a valid argument in favor of a mitochondrial fl-oxidation system in higher plant cells.

B. Palmitoylcarnitine-Dependent Oxygen Uptake In contrast to the mitochondrial fl-oxidation system of animal ceUs, the peroxisomal fl-oxidation system of both animal and plant ceUs is insensitive to KCN in the presence of NAD (Section IV.A.2). Higher plant peroxisomes also do not oxidize acylcarnitines (Section IV.H). Palmitoylcarnitine- (or palmitoyl-CoA plus L-carnitine)-dependent oxygen uptake by mitochondrial fractions from pea cotyledons has been observed repeatedly in the presence of sparker malate and under state 3 conditions, t°s'HS-jl8 Due to progress in the isolation of purified organelle preparations and since KCN-sensitivity of this oxygen uptake has been demonstrated only recently, m°sthe more recent publications on this topic will only be considered here (for former discussion see Ref. 5). The palmitoylcarnitinedependent oxygen uptake by mitochondrial fractions from pea cotyledons has been interpreted to demonstrate mitochondrial fl-oxidation. I°s,"7,11s For the reasons mentioned above, peroxisomes contaminating the mitochondrial fractions can be excluded as the site of the palmitoylcarnitine-dependent, KCN-sensitive oxygen uptake. Using similar assay conditions, palmitoylcarnitine-dependent, KCN-sensitive oxygen uptake by plant mitochondria (including pea cotyledon mitochondria) was not observed in other studies (Ref. 5; Gerhardt, unpublished data; but see below). Assuming the unlikely case that palmitoylcamitine was completely oxidized to COs and H20 via fl-oxidation and the tricarboxylic acid cycle, the observed palmitoylcarnitinedependent oxygen uptake (35 nmol 02" min l" mg ~) by the pea cotyledon mitochondria ~°8 corresponds to 1.5 nmol palmitoylcarnitine oxidized .rain I. mg -I. This amount is approximately five times higher than the total carnitine long-chain acyltransferase activity (0.3 nmol palmitoylcarnitine formed-min~.mg l) determined in pea cotyledon mitochondria (mitoplasts). 1°8 Of this activity, the intramitochondrial portion would only contribute to the conversion of palmitoylcarnitine to palmitoyl-CoA oxidized finally. Thus, the rate of palmitoylcarnitine formation was approximately 10-fold lower than the rate of presumed complete palmitoylcarnitine oxidation. I°s However, using similar assay conditions, a carnitine long-chain acyltransferase activity of pea cotyledon mitochondria (mitoplasts) has been reported since, "a which was approximately 50-fold higher than that reported earlier, l°s The rate of presumed palmitoylcarnitine oxidation was unchanged. Jm8 Assay mixtures used to demonstrate palmitoylcarnitine-dependent oxygen uptake by pea cotyledon mitochondria routinely contained sparker malate and thiamine pyrophosphate. I°s'j~8 Plant mitochondria readily oxidize malate without the necessity of removing oxaloacetate. H9 Due to the presence of NAD-dependent malic enzyme in plant mitochondria, citrate is formed at malate oxidation in the presence of thiamine pyrophosphate, a cofactor of pyruvate dehydrogenase.H9 Oxidation of sparker malate (50 #t,l) by potato tuber mitochondria was demonstrated using labelled substrate (Maier and Gerhardt, unpublished data). The oxidation was stimulated by palmitoylcarnitine. N A D H formation due to oxidation of sparker malate, malate or other substrates oxidized in a NAD-dependent reaction was greatly stimulated, under isoosmotic assay conditions, by palmitoylcarnitine at/tM concentrations in mitochondria from avocado (Persea americana L.) mesocarp or potato tubers (Wolf, Maier and Gerhardt, unpublished data). Palmitoyl-L-carnitine and palmitoyl-I)L-carnitine were equally effective while L-carnitine was ineffective. Mitoehondrial reactions depending on the mitochondrial electron transport chain were stimulated at low but inhibited at higher palmitoylcarnitine concentrations (#M range).

440

B. GERHARDT

The palmitoylcarnitine effects on mitochondrial reactions were similar to those obtained with Triton X-100 at different concentrations. They were reduced or abolished in the presence of bovine serum albumin. At certain ratios of palmitoylcarnitine to bovine serum albumin, palmitoylcarnitine-depedent oxygen uptake was observed with potato tuber mitochondria, provided that sparker malate was present in the assay mixture (Fischer and Gerhardt, unpublished data). For the first time, the palmitoylcarnitine-dependent oxygen uptake reported for pea cotyledon mitochondria could be confirmed. However, the preliminary data outlined above suggest that palmitoylcarnitine at/~M concentrations has a disintegrating effect on mitochondrial membranes, resulting in facilitated access of external substrates to intramitochondrial reaction sites and stimulation of reaction rates. Thus, palmitoylcarnitine-dependent oxygen uptake by mitochondria in the presence of sparker malate has to be interpreted with caution as evidence for mitochondrial 8oxidation activity. When potato tuber mitochondria were provided with palmitoyl-t~-carnitine labelled at the C-1 position of the fatty acid moiety, neither formation of (14C)acetyl-CoA nor that of labelled citrate, in the presence of sparker malate, nor loss of radioactivity from the substrate were detected (Maier and Gerhardt, unpublished data).

C. Enzymes of/~-Oxidation Activities of/~-oxidation enzymes were detected in mitochondrial fractions isolated from plant tissues on density gradients. 29-32,t~%~2°If these activities are not due to peroxisomal contamination, they can represent mitochondrial constituents, except acyl-CoA oxidase activity. So far, acyl-CoA oxidase is considered to be a peroxisomal marker enzyme. The first oxidation step at mitochondrial E-oxidation in animal cells is catalyzed by acyl-CoA dehydrogenase. Up to now, all attempts to demonstrate acyl-CoA dehydrogenase activity in higher plant mitochondria have been unsuccessful using long-, medium- and short-chain acyl-CoAs as substrates. 3°-33'39'm Acyl-CoA dehydrogenase does not seem to be inactivated during mitochondria isolation since the ratio of glycerol-3-phosphate dehydrogenase activity to acyl-CoA dehydrogenase activity of insect mitoehondria did not change when insect mitochondria were added to a homogenate from mung bean hypocotyls and reisolated with the plant mitochondria, m When the ratio of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase or thiolase activity to the activity of the peroxisomal marker catalase (or glycolate oxidase) was calculated for both mitochondrial and peroxisomal fractions from the same gradient, a statistically significant difference between the ratios obtained for mitochondrial and peroxisomal fractions did not result, except in one case concerning thiolase (see below).29'32 The/~-oxidation enzyme activities in mitochondrial fractions were therefore attributed to contaminating peroxisomes rather than to mitochondrial constituents. However, values of the above mentioned ratios higher in mitochondrial fractions than in peroxisomal fractions have also been reported and were interpreted to demonstrate true mitochondrial /~oxidation activity. "7'~2° This interpretation may however be problematic as indicated by two examples. (i) Mitochondrial fractions isolated from potato tubers exhibited a ratio of thiolase to catalase activity higher than the peroxisomal fractions. 32 But, thiolase activity exhibited hyperbolic distribution on the gradient (Gerhardt, unpublished data). This distribution indicated that thiolase leaked out very easily from its housing organeUe and resulted in thiolase activity higher in the mitochondrial than in the peroxisomal fraction. (ii) As an intact inner mitochondrial membrane is impermeable for acyl-CoAs, enzyme assays on mitochondrial and, in parallel, peroxisomal fractions were performed by Gerhardt 29'32 in the presence of 0.05% Triton X-100. Thomas and coworkers tMT'~2°who observed an inhibitory effect of 0.2% (!) Triton X-100 on 3-hydroxyacyl-CoA dehydrogenase .7 used a certain procedure to obtain ruptured mitochondria. The procedure performed on isolated mitochondria and peroxisomes in parallel included dilution, recentrifugation and subsequent osmotic breakage of the organelles (mitochondria). Data were not reported


441

on the loss of t-oxidation enzyme activities, mitochondrial marker enzyme activities and/or organelle protein following dilution and recentrifugation, especially with regard to the fragile peroxisomes and the well known differential loss of proteins from organelles. Applying the procedure used by Thomas and coworkers to mitochondrial fractions from sunflower cotyledons it was observed that the specific activity of peroxisomal acyl-CoA oxidase as well as the ratio of acyl-CoA oxidase to catalase activity in treated mitochondrial fractions were higher than in untreated mitochondrial fractions (Gerhardt and Fischer, unpublished data). Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity of the peroxisomal t-oxidation system are activities of the multifunctional protein (Section IV.A.l.c). In contrast, these activities belong to individual proteins in animal mitochondria. A protein exhibiting only enoyl-CoA hydratase activity and differing from the peroxisomal multifunctional protein in other properties too (thermostability; salt activation) has very recently been isolated from pea cotyledon mitochondria, m Antibodies raised against rat liver mitochondrial enoyl-CoA hydratase gave a positive signal in Western blots of total mitochondrial protein from pea cotyledons; no signal was observed when blots of total peroxisomal protein were probed. D. Conclusion

So far, the strongest argument in favor of mitochondrial//-oxidation in pea cotyledons appears to be the demonstration of a mitochondrial protein exhibiting only enoyl-CoA hydratase activity and differing also in other characteristics from the peroxisomal multifunctional protein.~22 However, the presence of an enzyme of a pathway is not proof that the pathway exists and functions. Direct demonstration of acetyl-CoA formation from palmitoylcarnitine by pea cotyledon mitochondria is primarily needed to establish the existence of a mitochondrial//-oxidation system. In addition, the enzyme catalyzing the first oxidation step of the SUlbposed mitochondrial t-oxidation has to be demonstrated since failure to detect acyl-CoA dehydrogenase has repeatedly been reported (Section V.C). Thus, the present answer to the question of whether higher plant mitochondria possess t-oxidation activity has still to be that the existence of a t-oxidation system in higher plant mitochondria has yet to be demonstrated unequivocally. VI. a - O X I D A T I O N OF F A T T Y ACIDS

Oxidation in the *t-position of the fatty acid carbon chain occurs at (formation and) degradation of 2-hydroxy fatty acids, intermediates at peroxisomal catabolism of certain fatty acids which are degraded by the//-oxidation pathway at first (Sections IV.B and IV.D.2.c). Following oxidation of 2-hydroxy fatty acids by 2-hydroxy acid oxidase, the 2-oxo acid formed undergoes oxidative decarboxylation leading back to the acyl-CoA track of//-oxidation. However, the term *t-oxidation actually refers to a different process located presumably at the endoplasmic reticulum, known to act only on free fatty acids of C~4:0to Cjs:x chain length and yielding a free fatty acid containing one carbon atom less than the parent fatty acid, CO2 and/or D-2-hydroxy fatty acid. According to the reaction mechanism proposed by Shine and Stumpf,67 the intermediate of this process is a D-2-hydroperoxyl fatty acid (C,; 14 < n < 18) which is decarboxylated to fatty aldehyde (C,_j) and/or reduced to v-2-hydroxy fatty acid (C,). The fatty aldehyde is oxidized by a NAD-dependent fatty aldehyde dehydrogenase, yielding a free fatty acid (Cn_ ~). The o-2-hydroxy fatty acid is considered to be a deadend product with respect to fatty acid degradation. However, degradation of v-2-hydroxy fatty acid by peroxisomes has been reported (Section IV.B). The reaction mechanism of ,t-oxidation proposed by Shine and Stumpfs7 and former proposals as well as possible physiological functions of at-oxidation were reviewed last in 1980.4 Since then, no substantial progress in our knowledge of both the reaction mechanism and the physiological function of *t-oxidation has been made.

442

B.

GERHARDT

The ~-oxidation process is thought to be localized at the endoplasmic reticulum but experimental evidence for that is scarce. Reoxidation of NADH generated at r,.oxidation could occur by the NADH dehydrogenase located on the outer face of the inner mitochondrial membrane. However, indirect evidence has been presented that NADH generated at ~-oxidation is reoxidized by a more complicated system, a malateoxaloacetate shuttle linking also fatty acid ~t-oxidation to the mitochondrial electron transport chain. ~23 Besides potato tubers, ~24 or-oxidation of fatty acids is presumably involved in initial wound respiration of those bulky storage organs which are characterized by KCN-sensitive wound respiration, low amounts of neutral lipids and high levels of linolenic acid (contained in galactolipids and/or phospholipids)) 9 The synthesis of odd-chain alkanes, components of plant surface waxes, from very long-chain even-numbered fatty acids does not involve or-oxidation. The alkanes are synthesized from acyl-CoAs which are reduced first to the corresponding aldehydes by an acyl-CoA reductase. The aldehydes subsequently undergo decarbonylation yielding alkanes and C O . 125'126 VII. DEGRADATION OF FATTY ALCOHOLS Substantial amounts of fatty alcohols are degraded in jojoba (Simmondsia chinensis (Link) Schneid.) cotyledons during postgerminative growth of the seedlings. ~27The fatty alcohols arise from hydrolysis of wax esters which as a form of storage lipid in higher plants are only known for jojoba seeds. The wax esters are localized in membrane-bound organelles (wax bodies) and hydrolyzed by an alkaline hydrolase.12s The carbon of the wax esters is directed into gluconeogenesis 127 following oxidation of the fatty alcohols to the corresponding fatty acids. Main constituents of the jojoba wax esters are eicosenoic acid (20:1, 11-cis), eicosenol and docosenol (22:1, 13-cis). t29"13° A fatty alcohol oxidizing system associated primarily with the wax body membrane has been demonstrated in jojoba cotyledons./3~ Molecular oxygtn served as electron acceptor at the oxidation of the fatty alcohols to fatty aldehydes. The 1:1 stoichiometry observed for oxygen uptake and fatty aldehyde formation suggests that oxygen is reduced to H202 by a fatty alcohol oxidase. The subsequent oxidation of fatty aldehydes to fatty acids is a NAD-dependent reaction. The fatty alcohol oxidizing system preferentially reacted on dodecanol. The physiological substrates eicosenol and docosenol were oxidized with approximately one-fifth of the rate observed with dodecanol as substrate. TM VIII. FATTY ACID DEGRADATION IN ALGAE Enzyme activities of the fl-oxidation reaction sequence have been demonstrated in algae of different evolutionary lines. Studies on subcellular localization of the fl-oxidation enzymes showed that the enzymes are localized in peroxisomes and/or mitochondria. The subcellular location of the enzymes in an alga presumably depends on the evolutionary line to which the alga belongs. Enzymes of fl-oxidation were detected in peroxisomes in Euglena (Euglenophyta) 132and the green algae Mougeotia (Zygnematales, Charophycea, Chlorophyta) ~aa't~ and Rhizoclonium (Siphonocladales, Ulvaphycea, Chlorophyta). 135 They were demonstrated in both peroxisomes and mitochondria in the unicellular green alga Eremosphaera (Chlorococcales, Chlorophycea, Chlorophyta) ~3a'~uand in members of the group of Micromonadophyceae/ Prasinophyceae (Chlorophyta) m which are thought to be the most primitive group of green algae, t3t'-~3s Mitochondria were shown to be the subcellular site of fl-oxidation enzymes in the Tribophycacean (Xanthophycean) algae Bumilleriopsis ~34'139and Vaucheria ~4°as well as in Cyanidium (Rhodophyta), ~4~ algae which do possess peroxisomes. Acyl-CoA synthetase demonstrated in mitochondria of Cyanidium appears to be a membrane-bound enzyme.t4~ However, whether the activity is located at the outer and/or inner mitochondrial membrane or to which face of the inner mitochondrial membrane the enzyme is bound were not studied. Carnitine long-chain acyltransferase activity was


443

detected neither in mitochondria of Cyanidium nor in mitochondria of Bumilleriopsis) 41 Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity of Cyanidium mitochondria could be separated from each other, 14~ indicating that the activities belong to two different proteins as in mammalian mitochondria. The acyl-CoA oxidizing enzyme of algal mitochondria showing B-oxidation enzyme activities appears to be an acyl-CoA dehydrogenase. ~'t35a4° In the presence of acyl-CoA, the mitochondria performed a phenazine methosulfate-mediated reduction of dichlorophenol indophenol but did not show oxygen uptake (if low oxygen uptake was observed it could be inhibited by KCN, indicating involvement of the respiratory chain). In contrast, algal peroxisomes possessing B-oxidation enzymes reduced oxygen in the presence of acyl-CoA although the electrons could also be transferred from acyl-CoA to diehlorophenol indophenol (a reaction not demonstrated for acyl-CoA oxidase of higher plant peroxisomes so far). '33-'35 Inhibitors (KCN, NAN3) of catalase did not influence the measurable oxygen uptake and concomitant H202 formation was not observed. ~34When the acyl-CoA-dependent oxygen uptake by algal peroxisomes was assayed under 1sO2, the isotope was detected in water rather than in H202. The results indicate that algal peroxisomes which exhibit B-oxidation enzyme activities possess an acyl-CoA oxidase which reduces oxygen to water rather than to H202 generated by the acyl-CoA oxidase of higher plant peroxisomes (Section IV.A. 1.b). Reduction of oxygen to water by algal peroxisomal acyl-CoA oxidase seems reasonable at least in peroxisomes of Micromonadophycean/Prasinophycean algae since catalase has been detected neither in cell-free extracts from these organisms nor in their peroxisomes) 35 Based on relative activities, the peroxisomal acyl-CoA oxidizing enzymes of Mougeotia and Eremosphaera and the mitochondrial acyl-CoA oxidizing enzyme of Bumilleriopsis exhibited highest activities towards medium- and/or long-chain acyl-CoAs.'34 The enzymes also showed Km values for palmitoyl-CoA (70-80/aM) lower than those for hexanoyl-CoA (100-150/aM). The mitochondrial acyl-CoA oxidizing enzyme of Eremosphaera exhibited preferential activity and affinity towards short-chain acyl-CoAs. However, more information is needed to answer the question, arising from the results, of whether the peroxisomal and mitochondrial B-oxidation systems complement each other in those algae (e.g. Eremosphaera) possessing both systems. The Chlorophyta are divided into several evolutionary lines thought, at present, to evolve from ancestors which were probably similar, in their structural characteristics, to members of the Micromonadophycean/Prasinophycean group.'36-'38 These algae possess both peroxisomal and mitochondrial B-oxidation systems.'35 Therefore, it may be speculated that the fate of the two B-oxidation systems has been different during evolution of the Chlorophytan lines. The higher plants evolved from the Charophycean line t36q3s and, a Mougeotia member of this line, possesses, and so has retained, the peroxisomal B-oxidation system only. However, the acyl-CoA oxidases of algal and higher plant peroxisomes appear to differ in their electron transfer characteristics (see above). How fundamental these differences are, can only be answered when information on the reaction mechanism and/or the amino acid sequence of the two acyl-CoA oxidases becomes available. Acknowledgement--The research on fatty acid degradation in the author's laboratory has beensupported by the Deutsche Forschungsgemeinschaft. (Received 23 June 1992)

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