Eur. J. Biochem. 57, 425-430 (1975)
Acetoin Degradation in Bacillus subtilis by Direct Oxidative Cleavage Juan M. LOPEZ, Brigitte THOMS, and Hartmut REHBEIN Arbeitsgruppc Biocheinie der Mikroorganismen, Abteilung fur Biologie, Ruhr-Univcrsitat Bochuiii (Received February 28/June 5 , 1975)
Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subtilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate. The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol. In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin-cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcdrbinol synthase, thought to be responsible for the formation of diacetylinethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.
Acetoin is a fermentation product excreted by many microorganisms when they grow in media containing glucose or other carbon sources which are degraded via the Embden-Meyerhof-pathway. It can be reutilized after glucose is exhausted, and therefore it may be considered as an energy-storing metabolite. The dissimilation of acetoin proceeds according to Juni and Heym [ l ] viu a cyclic degradative pathway : the 2,3-butanediol-cycle, which has acetate as the endproduct. Although Bucillus subtilis can degrade all intermediates of this cycle [2], a complete cycle is not essential for the dissimilation of acetoin. Mutants blocked in two of its reactions can metabolize acetoin as well as the standard strain [ 3 ] .This finding prompted us to investigate the possibility of an alternative pathway. I n this work it is demonstrated that B.subrilis cleaves acetoin directly into two molecules of acetaldehyde, one of which is in an activated form and is subsequently oxidized to acetate. MATERIALS AND METHODS Bacterial Struiizs All strains employed were derived from B. subtilis 168. The standard strain B. suhtilis 60015 requires -. -. ~
~
Enzjnze. Pyruvate dehydrogenase (EC 1.2.2.2).
Eur. J. Biochem. 57 (1975)
tryptophan and methionine for growth. Strain 61141, which is deficient in pyruvate dehydrogenase activity [4], requires in addition acetate. Strains 60174, requiring adenine and thiamine, and 6141 1, requiring uracil, were kindly provided by Dr E. Freese. The latter strain is deficient in acetoin dehydrogenase and fructose-1-phosphate kinase. Upon growth in glucose media this strain accumulates acetoin but does not reutilize it even after supplementation with uracil.
Media and Growth Conditions TBAB plates contained 33 g/l tryptose blood agar base (Difco). Nutrient sporulation medium [ 5 ] , consisted of 8 g/l nutrient broth (Difco), supplemented with 1 yM FeCl,, 0.7 mM CaCI,, 0.5 mM MnCI,, 20 mM K,HP04 and 45 mM KH,PO,. Donnellan medium [6] was supplemented with 0.1 casamino acids (Difco) or in case of strain 60174 with vitaminfree casamino acids (Difco), 10 yg/ml thiamine and 10 yglml adenine. Prewarined media were inoculated from overnight grown TBAB plate cultures. The cells were suspended in the fluid culture medium to an absorbance at 578 nm of about 0.1 and were shaken in conical flasks in a reciprocal water bath shaker at 100 strokesjmin at 37°C. The flasks were filled only up to 25 :d of their volume.
:(
426
PwImztioti (? f E.\-tr-act.\ '
Bacteriawereharvested by centrifuging at 40000 x g for 5 inin at 4 C. then washed twice and resuspended in 0.1 M potassium maleatc pH 6.5 and lysed in a French press at 5000 Ib in'. Cell debris were removed by centrifugation at 40000 x g for 30 inin at 4 C : the clear supernatant was used for enzyme tests 01- dissiinilation experiments.
Prrpai.(it iotI 0f PertI i c w b ilir c ~ Cl'clls '
Bacteria were washcd twice and suspended to a density of 2 x 10'" cellslinl in 0.2 M potassium phosphate buffer pH 7.0. 10 nil of the suspension were wat-med to 37 C , mixed with 0.5 ml toluene and incubated at 37 C for 10 inin. The cells were then washed twice and suspended in 1 in1 of the same buffer.
Dcjtuniinutioti of'Mt~tcrholitt~.r Acetoin was determined by the method of Westerfeld [7]: 0.5 in1 creatine (5 mglml) and 0.5 nil a-naphthene (50 1ng;inl in 2.5 N KOH) were added to the samples, containing less than 0.2 p o l acetoin in 2.5 ml H,O. The mixture was incubated at room temperature for 1 h. The absorbance was read at 540 nm against a blank. Acetyl phosphate was determined by the hydroxamic acid method [XI : 0.2ml 4 M hydroxylarnmonium chloride solution of pH 6.6 were added to 0.8 ml samples; after 10 inin at room temperature 1 nil of 10",, trichloroacetic acid and 4 ml of FeCI, . 6 H,O (12.5 mg/nil) in 1 N HCl were added. 5 inin later the absorbance at 500 nin was read against a blank. For the gas chroinatographic analysis a flame ionization detector and N2 as carrier gas was used. Acetoin and methylacetoin were separated in a glass column, 5 feet long and 1:'8 inch internal diameter, filled with 20)::, NPGS 2",, H,PO, on firebrick, 60/80 mesh; injector temperature : 195 C ;column temperature : 95 'C for acetoin and 80 "C for methylacetoin determination; detector temperature: 210 'C, flow rate: 25 ml N,/ inin; samples of 1 1-11 were injected. Diacetyl and its dissimilation products were separated in a glass column, 9 feet long and l / 8 inch internal diameter, filled with 12'5,, DEGS;A-SD 70180 mesh; injector temperature : 1 10 C ; column temperature : 110 C ; detector temperature: 150 ' C ; flow rate: 30 in1 N,/inin. A reaction mixture containing cell extract (10 mg protein,'ml), dichlorophenolindophenol (50 mg/ml), 0.1 M potassium inaleate pH 6.5 and as substrate diacetyl (5 pniol:ml), acetoin (25 pmo1:ml) or methylacetoin (25 pmol!ml) was incubated at 35 ' C for 60 min. Then 10 N HCI was added to obtain a pH of 1 - 2.After centrifugation of the protein precipitate an aliquot of
+
the supernatant was iii-jected for the deterinination of the degradation products. Protein determinations were done according to Low-y ct (11. [9].
E ~ I Z J VA.s.su~~.s ~IC Thc specific activity of acetoin dehydrogenase is exexpressed as dichlorophenolindophenol reduced i n pmol'niin per milligram protein at 25 'C. The assay mixture contained 100 pmol potassium phosphate pH 7.0,0.05 pniol thiamine pyrophosphate, 0.05 pmol NAD, 0.077 p o l diclilorophenolindoplieiiol and 4 pmol acetoin per ml. The same mixture was used for the pyruvate dehydrogenase test with acetoin replaced by pyruvate. The reduction of dichlorophenoliiidophenol was recorded at 578 mi. The reaction was started by substrate addition. Diacetylmethylcarbinol synthase activity was determined as described by Juni and Heym [lo].
Prepurcition c ~ f 4 - A n 1 i i 1 0 h ~ t ) ~ ~ ~ i l u ' t ~ l ~4~B~ 1 ~ ~ ~ - S ~ ~ ~
j;)r.tlic Hyu'roplplzohic~Clir.omatogr.rii~}i!. Sepharose 4 B was BrCN-activated as described by March Pt al. [l I ] . The coupling with 4-aminobutyraldehydediethylacetal was carried out as follows : 7 g of wet, activated Sepharose were mixed with a solution of 12 mmol acetal in 20 in1 0.2 M NaHCO, buffer pH 9.5 ; the suspension was stirred overnight at 5 "C and washed several times with buffer and water. Acetal hydrolysis was performed with 0.2 N HCI at 37 ' C for 30 inin. The determination of the liberated ethanol yielded 9 pinol bound aldehyde per g of wet Sepharose.
RESULTS A N D DISCUSSION
CIeavugc of Actjtoin, Diacriyl and Metliylacetoiii B. suhtilis cells dissimilate acetoin. Concomitantly cell extracts contain a dichlorophenolindophenolreducing activity with acetoin as substrate. This activity was induced by acetoin [2]. The mutant 61411, which is unable to degrade acetoin, lacks dichlorophenolindophenol-reducing activity (Fig. 1). If the mutant or the standard strain 60015 were grown in nutrient sporulation medium enriched with 10 mM glucose, acetoin accumulated. In contrast to the standard strain the mutant was unable to re-use it again. In order to identify the products which result froin the oxidation of acetoin in the presence of dichlorophenolindophenol,extracts from standard strain cells were incubated for 60 min at 3.5 C and aliquots of the mixture were then examined in the gas chromatograph. The results are shown in Table 1. Acetaldehyde Etir. J . Riochcin. -77 (1975)
I . bl. Lhper. 13. l'hoins, and H. Relibcin
427
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1.0 -
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35
3.5
30
3.0
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0.5 -
2 0.4n m
$ 0.3.~ m 25 < k
2.5 ='
. E
0.2 - g V
g 20
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-
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L -
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1
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0
c
0.1- ' $ 15
Acetoin Diacetyl Methyl-
107 234
87 0
0 0
acct oi 11
85
0
> 48
0
0
I '
2
1 .0
0.5
2
3
4
5
6
7
24
0
Time ( h )
Fig. 1. Acctoii? cleli~~rlr.o~[,ir~iI.ve ac/ir.itj. c m r l cicefoiii ufilisrrtioii iii €3. subtilis .straiiis 6001.5 wid 6/4/f.Strains 60015 (standard strain) and 6141 1 (acetoin tiehydrogenasc- )in nutricnl sporulation medium cni-ichcd with 10 mM glucosc. At different times aliquots of thc cultui-cs were taken and centrifuged. Acetoin was deterinincd in the supernatant and acetoin dchydrogcnase in the extracts of the centi-ifugdtcd cells. (0 0 ) Absorbance at 578 nm of both culturcs: (O----O) acetoin concentration in the culture fluid of strain 6141 1 (AcDIJ-); (O----O) acetoin dehydrogen activity in the strain 61411 (AcDH..) cell extracts: ( O - - -0)acetoin concentration i n thc culture fluid of strain 60015; (W--W) acetoin dehydrogenase specific activity in the strain 6001 5 cell extracts -
I n order to support either of these pathways the degradation of diacetyl was measured. As Table 1 shows no acetaldehyde was found, i.e. diacetyl was completely oxidized to acetate. This result suggests a direct splitting of acetoin, without its previous oxidation to diacetyl, followed by the oxidation of acetaldehyde. If this was true, one would expect that 2-hydroxy-2-methyl-butanone-3 (inethylacetoin) also could be cleaved in a similar fashion although this compound can not be oxidized to a diketone, because it is a tertiary alcohol. Cell extracts actually catalyze the degradation of inethylacetoin in the presence of dichlorophenolindophenol. Table 1 shows that acetatc and acetone are formed, suggesting the following reaction scheme:
CH3
I
and acetate were found in comparable amounts, whereas no diacetyl could be detected. There are two alternative oxidation sequences : a) Acetoin is oxidized to diacetyl. This is subsequently cleaved to acetaldehyde and acetate. The cleavage is so fast that no diacetyl can be detected. b) Acetoin is directly cleaved into two molecules acetaldehyde and a fraction of the aldehyde is subscquently oxidized to acetate by dichlorophenolindophenol (C1,Ind). These alternatives can be described as follows:
a) H,C-C-CH-CH,
I/
I
C1,lod Cl,lnd
II
I
kur J Biochem 57 (1975)
CI,Ind Cl,lnd
H,C-C -CH,
II
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OH 0
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+
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0 0
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'OH
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+
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H
-
H,C-C
-
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< I2lnd H7
0
//
\
OH
+ H,C-C
0 // \
H
.
Acetoin Degradation in BuciNus suhtilis
428
In order to prove whether this direct oxidative degradation pathway is also used in vivo, the following experiment was carried out: mutant 61 141, lacking pyruvate dehydrogenase activity and therefore being unable to synthesize acetyl-CoA in the absence of acetate, was grown in Donnellan medium. In this medium the strain stops growth at a density of 2 x 10’ cells/ml. Growth can be restored, however, by adding compounds able to be metabolized to acetate and of course by acetate itself. If methylacetoin would restore growth, one can conclude that the cell is able to perform the former reaction, e.g. to split the molecule without the previous formation of the diketone. This is in fact the case (Fig. 2). The growth rate with methylacetoin is not as high as that obtained with acetoin. This is expected, since two molecules of acetate can be formed by the dissimilation of acetoin, but only one molecule of acetate is produced from methylacetoin. The acetone that is simultaneously produced can not be dissimilated by B. subtilis and is excreted into the medium (not shown). Therefore we propose that acetoin is split without its previous oxidation. This reaction gives rise to two molecules acetaldehyde which subsequently are partially oxidized. We will designate the enzyme(s) catalyzing this reaction as acetoin dehydrogenase. Diacetyl also can be split. In this case the reaction results in one molecule acetaldehyde and one molecule acetate. Thiamine Pyrophosplzate is Required,for the Cleavage The question arises why only about half of the acetaldehyde formed froin acetoin is further oxidized to acetate, whereas no acetaldehyde can be detected in the experiments froin diacetyl and methylacetoin. The cleavage of acyloin compounds and the oxidative decarboxylation of 2-oxoacids depend on the presence of thiamine pyrophosphate. This suggests that a thiamine-pyrophosphate-boundmolecule is an intermediate product in the acetoin degradation. Thiamine pyrophosphate would combine with the carbonyl group of the acetoin molecule forming the intermediate compound 2,3-butanediol-thiamine pyrophosphate. In a reaction analogous to the decarboxylation of pyruvate, the splitting of this molecule would produce two molecules acetaldehyde, one of which as hydroxyethylthiamine pyrophosphate. Hydroxyethylthiainine pyrophosphate can be readily oxidized by dichlorophenolindophenol [12]. The cleavage of diacetyl and methylacetoin produces no free acetaldehyde but acetate or acetone from the non-thiamine pyrophosphate-bound half molecule. In fact the oxidation of acetoin depends on thiamine pyrophosphate as the next experiment shows. Two cultures of the strain 60174, a thiamine auxotroph, were grown in Donnellan medium supplemented with
2.0
F
53 10rnM Acetoin 10 rnM Methylacetoin
No addition
0.1 -
0
1
2
3
4
5
Time (h)
Fig. 2. Acctoin and n~c~th~lcrcc~~oin irtilisutiorz in 111c struiri 6 I141 (lucking pqwvute dehydrogenuse) . Strain 61 141 (pyruvate dehydrogenase-) was grown in Donnellan medium enriched with 0.1”” casamino acids until growth stopped. Then the culture was divided and respectively acetoin, methylacetoin or nothing was added and growth was following afterwards. ( 0 0 ) Acetoin: (A- -A) inethylacetoin; (ap-.) no addition
Table 2. EjTccftcrof’tkiamine pyrophosphute on the ovidntion of ncefoiti Strain 60174 (thiamine-) was grown in Donnellan medium 0.1 vitamin-free casamino acids and 20 mM acetoin. Two cultures were grown, one of them suppleinented with 10 pgjml thiamine. When both cultures had reached a density of 5 x lo7 cellslinl they wcre harvested and acetoin dehydrogcnase, diacetylmethylcarbinol synthase and pyruvate dehydrogenase activities werc determined in the cell-free extracts with and without thiamine pyrophosphate i n the test mixture
+
Growth conditions
Assay conditions
Pyruvate dehydrogenasc activity
Diacetylmethylcarbinol synthase activity
100
100
100
100
100
100
Acetoin dehydrogenase activity
% + Thiamine + rhiainine Thiamine -
Thiamine
+ thiamine pyrophosphate - thiamine pyrophosphate + thiamine pyrophosphate - thiamine pyrophosphate
44.5 0
170 0
52.4 0
vitamin-free casamino acids and 20 mM acetoin, with and without thiamine. No acetoin dehydrogenase activity was found in the extract of the culture grown without thiamine if the assay was performed without thiamine pyrophosphate, as can be seen from Table 2. In the presence of thiamine pyrophosphate, however, about 45% of the activity of the thiamine-grown culture was obtained. In the extracts of cells grown Eur J Blochem 57 (1975)
J. M. Lopez, B. Thorns, and H. Rehbein
429
L
0
1
2 Time ( h)
Fig. 3. Acetoin dehydrogenase and diacetylinetk~lcarbinolsynthase activities in the strains 60015 and 61411 grown in Donnellan medium + 0.1 casamino acids after induction with 20 mM acetoin. (.-Acetoin --dehydrogenase .) activity in strain 60015; (00 ) diacetylmethyldiacetylmethylcarbinol syncarbinol synthase activity in strain 60015; (W- -0)acetoin dehydrogenase activity in strain 6141 1: (O--O) thase activity in strain 61411 ~
Table 3. E.xperiment to see if uctirnted acetate urises as a product .from acetoin di.rsimilation Toluenized cells, prepared as described in Materials and Methods. were incubated with the substrates (20 mM) at 30°C for 60 inin and the formed acetyl phosphate was determined. i n addition the mixture contained: potassium phosphate buffer pH 7.0 (80 mM), NH,CI ( 5 mM), MgSOd (0.1 mM), thiamine pyrophosphate (0.025 inM), N A D (1 mM). CoA (0.1 mM), L-cystcine (1 mM), phorpliot~ansacetylase(1 U inl) Substrate
No substrate Acetoin Diacetyl Methylacetoin Acetaldehyde Acetate
FOI-mectacetyl phosphatc ninolxml~’x1i~’ < 0.01 11.3 17.9 14.0 < 0.01 < 0.01
in the presence of thiamine no stimulation of the acetoin dehydrogenase activity was obtained by the addition of thiamine pyrophosphate, indicating the presence of sufficient amounts of endogeneous thiamine pyrophosphate. Similar results were obtained for pyruvate dehydrogenase (Table 2). Activated Acetate Arises as a Product porn the Acetoin Dissimilation The postulated analogy between the pyruvate and acetoin cleavage suggests that an activated acetate, such as acetyl-CoA or acetyl phosphate is the final product from the oxidation of hydroxyethylthiamine pyrophosphate rather than the free acetate. In an attempt to clarify this problem, washed, toluenetreated cells were incubated with acetoin, methylacetoin or diacetyl in the presence of CoA and phosphotransacetylase. The acetylphosphate produced was determined (Table 3). It can be seen that acetoin or its analogs give rise to acetyiphosphate. A direct Eur J Biochem. 57 I 1975)
~
activation of acetate or acetaldehyde was excluded, since no acetylphosphate was formed in controls containing these compounds. These experiments, however, do not allow a discrimination between the formation of acetyl-CoA and acetylphosphate. Therefore a preliminary sequence of reactions is proposed :
+ (Electron acceptor),, + x Acetaldehyde + (Electron acceptor)red + Acetoin =
Thlamlnr pyrup””’’”’
’“
t
Acetyl-X
CoAS or P.
The acetaldehyde produced froin acetoin can be further metabolized as suggested by the observation that B. subtilis is able to oxidate free acetaldehyde and to utilize it to make acetate (unpublished results from Juan Lopez and Volker Wiechern). Relation between Diacetl,lmethylcarbii~ol Synthase and Acetoin Dehjdrogenase Activities In the 2,3-butanediol cycle diacetylmethylcarbinol is formed from diacetyl [l].As we have found, diacetylmethylcarbinol was produced from diacetyl only in the absence of dichlorophenolindophenol (data not shown). This indicates that the transfer of hydroxyethyl thiamine pyrophosphate to a molecule of diacetyl occurs only in the absence of a proper electron acceptor. An analogous result was obtained by Juni and Heyin [13]. They detected diacetylmethylcarbinol after incubation of diacetyl with pyruvic oxidase under anaerobic conditions. The formation of diacetylmethylcarbinol may therefore not depend on the presence of a specific diacetylmethylcarbinol synthase but rather be a unspecific side reaction which also is catalyzed by acetoin dehydrogenase. If diacetylmethylcarbinol formation and cleavage of acetoin are catalyzed by acetoin dehydrogenase, diacetylmethylcarbinol synthase activity should be defective in mutant 61411 (acetoin dehydrogenase-). This expectation was fulfilled (Fig. 3).
430
J . M . Lopez. H. Thoins, and H . Rehbein: Acetoin Dcgradation in Boc~i//i/.s .srhfi/i.\
Fraction number Fig. 4. I/~.t/rop/rohrcc / r ~ f ) ~ i ~ f / f f ~ , ~ /or1 , t i / 4~-/i ir j/ i. i i i i o h r i l ~ . ~ ~ r ~ / t / c I , ! ~ t / ( ~ - . \ c ~B. / ~ / iI c rin1 r ~ ~ .of v c ~cell-free extract (5 111%prolein) w a s applied to the coluinn ( d = 16 iiim. 1 = 60 inin). The coluinn was gradually eluted with 100 in1 buffer 1 (10 mM sodium citrate pH 6.6, 5 inM MgSO,. 1 mM EIITA. 0.1 mM thiamine p)rophosphatc), thcn Mith 5 0 in1 buffei- 2 (hull'cr 1 with 50 mM NaCl) and then with 50 in1 bull'cr 3 (bufyer 1 with 1 hl Nac'l). Fractions of 5 1111 wci-c collected and tllc activities of acetoin dehydrogenase and d i ~ ~ c e t y l i ~ l e t l ~ y l c ; ~ r b i ~ i ~ ~ l s y t h a c c were deteriiiincd. (. . . . . .) Ultt-aviolct abwrption; (0- 0 ) dincct~linctliylc~irbinol synthase activity.inl; (0---o)acetoin deli) drogenasc activit) ml: the arrows indicalc the point of bufcr change
Since diacetylmethylcarbinol is in addition thiamine-pyrophosphate-dependent (Table 2) and since both activities elute together and at the same ratio from a hydrophobic chromatography column (Fig. 4) we tentatively assume that both reactions are catalyzed by the same enzyme. The significance of thc 2,3-butanediol cyclc is an open question also because until now one reaction of the cycle, the enzymatic oxidation of acetoin to diacetyl, could not be demonstrated. Diez et a / . [14] and Gabriel et a/. [15] reported that the reduction of diacetyl to acetoin is practically irreversible. Bryn ct a/. [16] found that the enzyme diacetyl (acetoin) reductase is not able to catalyze the oxidation of acetoin to diacetyl. Other attcmpts to detect this activity with extracts of different microorganisms also where unsuccessful [17- 191. Only Sebek and Randless [20] reported that cells of Pscudomonas flziorescens produce diacetyl if grown with acetoin or 2,3-butanediol. The dichlorophenolindophcnol reduction in B. s u / ~ tilis extracts does not run at the expense of the oxidation of acetoin to diacetyl as was previously proposed [ 3 ] The . direct cleavage of acetoin explains its dissimilation without the necessity to assume its oxidation to diacetyl. Further studies concerning the detailed mechanism of the degradation of acetoin and analogous compounds can be attempted after the purification of acetoin dehydrogenase, which is in progress. This work w a s supported i n part by a grant from the Dwt.sc.lir Forsc/~~irig.s~c~ir,irc~iir.sc~lrafi. We thank Dr Thauer for instructions in handling thc gas chromatograph. Part of this work is submitted
by H . Rchbein in partial fulfilment of the requirements for the Dr rer. nat. degree in the Abteilung fiir Chcmie, Ruhr-Universitlit Bochum.
REFERENCES 1. JLini, E. & Heym, G . A. (1956) J . Buctrriol. 71, 425-432. 7 LOpez. J. & Fortnagel, P. (1972) Bkdzim. BiopIij,.~.A(,fu.279. -. 554- 560. 3. LbpeL, J., Thorns, B. & Fortnagel. P. (1973) Eur. J . Biochrrii. 40,479-483. 4. Freese, E. & Fortnagel, U . (1969) J . Bucteriol. 99, 745-756. 5. Frccse, E. & Fortnagel. P. (1967) .I. Bacterial. 94. 1957- 1969. 6. Donnellan. I. E., Nags. E. H . & Levinson, H. S. (1964) J . Buctcviol. 87, 332- 336. 7. Westerfeld, W. W. (1945) J . B i d . Chcm. 161. 495-502. 8. Lipmann, F. & Tuttlc, C. (1945) J . Biol. Chem. 159, 21-28. 9. Lowry, 0. H.. Rosebrough, M . J., Farr, A. L. & Randell, R. J. (1951) J . Biol. c'/imni. 193, 265. 10. Juni, E. & Hcym. G. A. (1956) J . Bacteiiol, 72, 746-753. 11 March. S. C., Parikh & Cuatrecasas. P. (1974) Anal. Bioc./wrn.60, 149- 152. 12. Holrcr, H. & Goedde, H. W. (1957) Biochrm. 2.320,192-208. 13. Juni, E. & Heyin, G. A. (1956) J . Biol. Clrem. 218. 365-378. 14. Diez. V . , Burgos. J . & Martin. R . (1974) Bioc~liini. Bioplrj..\. Acta. 350. 253 262. 15. Gabriel, M. A,,Jabara, H. & Al-Khalidi, U . A. S. (1971) Bioclirm. J , 124, 793- 800. 16. Bryn. K., Hctland, 0. & Stormer, F. C. (1971) Eur. J . Biocliem. 18, 116-119. 17. Seitz, E. W.. Sandive, W. E., Ellitzer. P. R . & Day, E. A. (1963) Crm. J . Microhid. 9, 431 -441. 18. Strecker, H. J. & Harary, I. (1954) J . B i d . C h m . 211,263-270. 19. Suoinalanen, H. & Linnahaline (1966) Arch. Bioc~herii.Biop/rj..c. 114, 502-513. 20. Sebek, 0. K. & Randlcs, C. I. (1952) Arch. Bioc~he,un. Biophj,.s. 40. 373 - 379. -
J . M . Lbpcz. B. Thorns. and H. Rehbein, Arbcitsgruppe Bioclieinie der Mikroorganismen, Abteilung fur Biologie der Ruhr-Universitiit Bochum. D-4630 Boclium-Querenburg. Poslracli 21 48, Federal Republic of Germany
Eur. J. Biocheni. 57 (1975)
Acetoin Degradation in Bacillus subtilis by Direct Oxidative Cleavage Juan M. LOPEZ, Brigitte THOMS, and Hartmut REHBEIN Arbeitsgruppc Biocheinie der Mikroorganismen, Abteilung fur Biologie, Ruhr-Univcrsitat Bochuiii (Received February 28/June 5 , 1975)
Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subtilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate. The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol. In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin-cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcdrbinol synthase, thought to be responsible for the formation of diacetylinethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.
Acetoin is a fermentation product excreted by many microorganisms when they grow in media containing glucose or other carbon sources which are degraded via the Embden-Meyerhof-pathway. It can be reutilized after glucose is exhausted, and therefore it may be considered as an energy-storing metabolite. The dissimilation of acetoin proceeds according to Juni and Heym [ l ] viu a cyclic degradative pathway : the 2,3-butanediol-cycle, which has acetate as the endproduct. Although Bucillus subtilis can degrade all intermediates of this cycle [2], a complete cycle is not essential for the dissimilation of acetoin. Mutants blocked in two of its reactions can metabolize acetoin as well as the standard strain [ 3 ] .This finding prompted us to investigate the possibility of an alternative pathway. I n this work it is demonstrated that B.subrilis cleaves acetoin directly into two molecules of acetaldehyde, one of which is in an activated form and is subsequently oxidized to acetate. MATERIALS AND METHODS Bacterial Struiizs All strains employed were derived from B. subtilis 168. The standard strain B. suhtilis 60015 requires -. -. ~
~
Enzjnze. Pyruvate dehydrogenase (EC 1.2.2.2).
Eur. J. Biochem. 57 (1975)
tryptophan and methionine for growth. Strain 61141, which is deficient in pyruvate dehydrogenase activity [4], requires in addition acetate. Strains 60174, requiring adenine and thiamine, and 6141 1, requiring uracil, were kindly provided by Dr E. Freese. The latter strain is deficient in acetoin dehydrogenase and fructose-1-phosphate kinase. Upon growth in glucose media this strain accumulates acetoin but does not reutilize it even after supplementation with uracil.
Media and Growth Conditions TBAB plates contained 33 g/l tryptose blood agar base (Difco). Nutrient sporulation medium [ 5 ] , consisted of 8 g/l nutrient broth (Difco), supplemented with 1 yM FeCl,, 0.7 mM CaCI,, 0.5 mM MnCI,, 20 mM K,HP04 and 45 mM KH,PO,. Donnellan medium [6] was supplemented with 0.1 casamino acids (Difco) or in case of strain 60174 with vitaminfree casamino acids (Difco), 10 yg/ml thiamine and 10 yglml adenine. Prewarined media were inoculated from overnight grown TBAB plate cultures. The cells were suspended in the fluid culture medium to an absorbance at 578 nm of about 0.1 and were shaken in conical flasks in a reciprocal water bath shaker at 100 strokesjmin at 37°C. The flasks were filled only up to 25 :d of their volume.
:(
426
PwImztioti (? f E.\-tr-act.\ '
Bacteriawereharvested by centrifuging at 40000 x g for 5 inin at 4 C. then washed twice and resuspended in 0.1 M potassium maleatc pH 6.5 and lysed in a French press at 5000 Ib in'. Cell debris were removed by centrifugation at 40000 x g for 30 inin at 4 C : the clear supernatant was used for enzyme tests 01- dissiinilation experiments.
Prrpai.(it iotI 0f PertI i c w b ilir c ~ Cl'clls '
Bacteria were washcd twice and suspended to a density of 2 x 10'" cellslinl in 0.2 M potassium phosphate buffer pH 7.0. 10 nil of the suspension were wat-med to 37 C , mixed with 0.5 ml toluene and incubated at 37 C for 10 inin. The cells were then washed twice and suspended in 1 in1 of the same buffer.
Dcjtuniinutioti of'Mt~tcrholitt~.r Acetoin was determined by the method of Westerfeld [7]: 0.5 in1 creatine (5 mglml) and 0.5 nil a-naphthene (50 1ng;inl in 2.5 N KOH) were added to the samples, containing less than 0.2 p o l acetoin in 2.5 ml H,O. The mixture was incubated at room temperature for 1 h. The absorbance was read at 540 nm against a blank. Acetyl phosphate was determined by the hydroxamic acid method [XI : 0.2ml 4 M hydroxylarnmonium chloride solution of pH 6.6 were added to 0.8 ml samples; after 10 inin at room temperature 1 nil of 10",, trichloroacetic acid and 4 ml of FeCI, . 6 H,O (12.5 mg/nil) in 1 N HCl were added. 5 inin later the absorbance at 500 nin was read against a blank. For the gas chroinatographic analysis a flame ionization detector and N2 as carrier gas was used. Acetoin and methylacetoin were separated in a glass column, 5 feet long and 1:'8 inch internal diameter, filled with 20)::, NPGS 2",, H,PO, on firebrick, 60/80 mesh; injector temperature : 195 C ;column temperature : 95 'C for acetoin and 80 "C for methylacetoin determination; detector temperature: 210 'C, flow rate: 25 ml N,/ inin; samples of 1 1-11 were injected. Diacetyl and its dissimilation products were separated in a glass column, 9 feet long and l / 8 inch internal diameter, filled with 12'5,, DEGS;A-SD 70180 mesh; injector temperature : 1 10 C ; column temperature : 110 C ; detector temperature: 150 ' C ; flow rate: 30 in1 N,/inin. A reaction mixture containing cell extract (10 mg protein,'ml), dichlorophenolindophenol (50 mg/ml), 0.1 M potassium inaleate pH 6.5 and as substrate diacetyl (5 pniol:ml), acetoin (25 pmo1:ml) or methylacetoin (25 pmol!ml) was incubated at 35 ' C for 60 min. Then 10 N HCI was added to obtain a pH of 1 - 2.After centrifugation of the protein precipitate an aliquot of
+
the supernatant was iii-jected for the deterinination of the degradation products. Protein determinations were done according to Low-y ct (11. [9].
E ~ I Z J VA.s.su~~.s ~IC Thc specific activity of acetoin dehydrogenase is exexpressed as dichlorophenolindophenol reduced i n pmol'niin per milligram protein at 25 'C. The assay mixture contained 100 pmol potassium phosphate pH 7.0,0.05 pniol thiamine pyrophosphate, 0.05 pmol NAD, 0.077 p o l diclilorophenolindoplieiiol and 4 pmol acetoin per ml. The same mixture was used for the pyruvate dehydrogenase test with acetoin replaced by pyruvate. The reduction of dichlorophenoliiidophenol was recorded at 578 mi. The reaction was started by substrate addition. Diacetylmethylcarbinol synthase activity was determined as described by Juni and Heym [lo].
Prepurcition c ~ f 4 - A n 1 i i 1 0 h ~ t ) ~ ~ ~ i l u ' t ~ l ~4~B~ 1 ~ ~ ~ - S ~ ~ ~
j;)r.tlic Hyu'roplplzohic~Clir.omatogr.rii~}i!. Sepharose 4 B was BrCN-activated as described by March Pt al. [l I ] . The coupling with 4-aminobutyraldehydediethylacetal was carried out as follows : 7 g of wet, activated Sepharose were mixed with a solution of 12 mmol acetal in 20 in1 0.2 M NaHCO, buffer pH 9.5 ; the suspension was stirred overnight at 5 "C and washed several times with buffer and water. Acetal hydrolysis was performed with 0.2 N HCI at 37 ' C for 30 inin. The determination of the liberated ethanol yielded 9 pinol bound aldehyde per g of wet Sepharose.
RESULTS A N D DISCUSSION
CIeavugc of Actjtoin, Diacriyl and Metliylacetoiii B. suhtilis cells dissimilate acetoin. Concomitantly cell extracts contain a dichlorophenolindophenolreducing activity with acetoin as substrate. This activity was induced by acetoin [2]. The mutant 61411, which is unable to degrade acetoin, lacks dichlorophenolindophenol-reducing activity (Fig. 1). If the mutant or the standard strain 60015 were grown in nutrient sporulation medium enriched with 10 mM glucose, acetoin accumulated. In contrast to the standard strain the mutant was unable to re-use it again. In order to identify the products which result froin the oxidation of acetoin in the presence of dichlorophenolindophenol,extracts from standard strain cells were incubated for 60 min at 3.5 C and aliquots of the mixture were then examined in the gas chromatograph. The results are shown in Table 1. Acetaldehyde Etir. J . Riochcin. -77 (1975)
I . bl. Lhper. 13. l'hoins, and H. Relibcin
427
2.0t
1.0 -
$ U> c
a
1
35
3.5
30
3.0
0.7-
0.5 -
2 0.4n m
$ 0.3.~ m 25 < k
2.5 ='
. E
0.2 - g V
g 20
2.0
U
22
-
3
L -
L
1
2
0
c
0.1- ' $ 15
Acetoin Diacetyl Methyl-
107 234
87 0
0 0
acct oi 11
85
0
> 48
0
0
I '
2
1 .0
0.5
2
3
4
5
6
7
24
0
Time ( h )
Fig. 1. Acctoii? cleli~~rlr.o~[,ir~iI.ve ac/ir.itj. c m r l cicefoiii ufilisrrtioii iii €3. subtilis .straiiis 6001.5 wid 6/4/f.Strains 60015 (standard strain) and 6141 1 (acetoin tiehydrogenasc- )in nutricnl sporulation medium cni-ichcd with 10 mM glucosc. At different times aliquots of thc cultui-cs were taken and centrifuged. Acetoin was deterinincd in the supernatant and acetoin dchydrogcnase in the extracts of the centi-ifugdtcd cells. (0 0 ) Absorbance at 578 nm of both culturcs: (O----O) acetoin concentration in the culture fluid of strain 6141 1 (AcDIJ-); (O----O) acetoin dehydrogen activity in the strain 61411 (AcDH..) cell extracts: ( O - - -0)acetoin concentration i n thc culture fluid of strain 60015; (W--W) acetoin dehydrogenase specific activity in the strain 6001 5 cell extracts -
I n order to support either of these pathways the degradation of diacetyl was measured. As Table 1 shows no acetaldehyde was found, i.e. diacetyl was completely oxidized to acetate. This result suggests a direct splitting of acetoin, without its previous oxidation to diacetyl, followed by the oxidation of acetaldehyde. If this was true, one would expect that 2-hydroxy-2-methyl-butanone-3 (inethylacetoin) also could be cleaved in a similar fashion although this compound can not be oxidized to a diketone, because it is a tertiary alcohol. Cell extracts actually catalyze the degradation of inethylacetoin in the presence of dichlorophenolindophenol. Table 1 shows that acetatc and acetone are formed, suggesting the following reaction scheme:
CH3
I
and acetate were found in comparable amounts, whereas no diacetyl could be detected. There are two alternative oxidation sequences : a) Acetoin is oxidized to diacetyl. This is subsequently cleaved to acetaldehyde and acetate. The cleavage is so fast that no diacetyl can be detected. b) Acetoin is directly cleaved into two molecules acetaldehyde and a fraction of the aldehyde is subscquently oxidized to acetate by dichlorophenolindophenol (C1,Ind). These alternatives can be described as follows:
a) H,C-C-CH-CH,
I/
I
C1,lod Cl,lnd
II
I
kur J Biochem 57 (1975)
CI,Ind Cl,lnd
H,C-C -CH,
II
H,
OH 0
+ H3C-C
+
// \
0 0
OH
H,C-C
I1 II
H,
C
H0
+ H,C-C,
'OH
H
0 0
0
0 OH
II
I
H,C-C-C-CH,
+ --
0 OH
b) H,C-C-CH-CH,
H,C- C - C- CH3-A+
+
2 H3C-C
//
\
H
-
H,C-C
-
fl,lnd
< I2lnd H7
0
//
\
OH
+ H,C-C
0 // \
H
.
Acetoin Degradation in BuciNus suhtilis
428
In order to prove whether this direct oxidative degradation pathway is also used in vivo, the following experiment was carried out: mutant 61 141, lacking pyruvate dehydrogenase activity and therefore being unable to synthesize acetyl-CoA in the absence of acetate, was grown in Donnellan medium. In this medium the strain stops growth at a density of 2 x 10’ cells/ml. Growth can be restored, however, by adding compounds able to be metabolized to acetate and of course by acetate itself. If methylacetoin would restore growth, one can conclude that the cell is able to perform the former reaction, e.g. to split the molecule without the previous formation of the diketone. This is in fact the case (Fig. 2). The growth rate with methylacetoin is not as high as that obtained with acetoin. This is expected, since two molecules of acetate can be formed by the dissimilation of acetoin, but only one molecule of acetate is produced from methylacetoin. The acetone that is simultaneously produced can not be dissimilated by B. subtilis and is excreted into the medium (not shown). Therefore we propose that acetoin is split without its previous oxidation. This reaction gives rise to two molecules acetaldehyde which subsequently are partially oxidized. We will designate the enzyme(s) catalyzing this reaction as acetoin dehydrogenase. Diacetyl also can be split. In this case the reaction results in one molecule acetaldehyde and one molecule acetate. Thiamine Pyrophosplzate is Required,for the Cleavage The question arises why only about half of the acetaldehyde formed froin acetoin is further oxidized to acetate, whereas no acetaldehyde can be detected in the experiments froin diacetyl and methylacetoin. The cleavage of acyloin compounds and the oxidative decarboxylation of 2-oxoacids depend on the presence of thiamine pyrophosphate. This suggests that a thiamine-pyrophosphate-boundmolecule is an intermediate product in the acetoin degradation. Thiamine pyrophosphate would combine with the carbonyl group of the acetoin molecule forming the intermediate compound 2,3-butanediol-thiamine pyrophosphate. In a reaction analogous to the decarboxylation of pyruvate, the splitting of this molecule would produce two molecules acetaldehyde, one of which as hydroxyethylthiamine pyrophosphate. Hydroxyethylthiainine pyrophosphate can be readily oxidized by dichlorophenolindophenol [12]. The cleavage of diacetyl and methylacetoin produces no free acetaldehyde but acetate or acetone from the non-thiamine pyrophosphate-bound half molecule. In fact the oxidation of acetoin depends on thiamine pyrophosphate as the next experiment shows. Two cultures of the strain 60174, a thiamine auxotroph, were grown in Donnellan medium supplemented with
2.0
F
53 10rnM Acetoin 10 rnM Methylacetoin
No addition
0.1 -
0
1
2
3
4
5
Time (h)
Fig. 2. Acctoin and n~c~th~lcrcc~~oin irtilisutiorz in 111c struiri 6 I141 (lucking pqwvute dehydrogenuse) . Strain 61 141 (pyruvate dehydrogenase-) was grown in Donnellan medium enriched with 0.1”” casamino acids until growth stopped. Then the culture was divided and respectively acetoin, methylacetoin or nothing was added and growth was following afterwards. ( 0 0 ) Acetoin: (A- -A) inethylacetoin; (ap-.) no addition
Table 2. EjTccftcrof’tkiamine pyrophosphute on the ovidntion of ncefoiti Strain 60174 (thiamine-) was grown in Donnellan medium 0.1 vitamin-free casamino acids and 20 mM acetoin. Two cultures were grown, one of them suppleinented with 10 pgjml thiamine. When both cultures had reached a density of 5 x lo7 cellslinl they wcre harvested and acetoin dehydrogcnase, diacetylmethylcarbinol synthase and pyruvate dehydrogenase activities werc determined in the cell-free extracts with and without thiamine pyrophosphate i n the test mixture
+
Growth conditions
Assay conditions
Pyruvate dehydrogenasc activity
Diacetylmethylcarbinol synthase activity
100
100
100
100
100
100
Acetoin dehydrogenase activity
% + Thiamine + rhiainine Thiamine -
Thiamine
+ thiamine pyrophosphate - thiamine pyrophosphate + thiamine pyrophosphate - thiamine pyrophosphate
44.5 0
170 0
52.4 0
vitamin-free casamino acids and 20 mM acetoin, with and without thiamine. No acetoin dehydrogenase activity was found in the extract of the culture grown without thiamine if the assay was performed without thiamine pyrophosphate, as can be seen from Table 2. In the presence of thiamine pyrophosphate, however, about 45% of the activity of the thiamine-grown culture was obtained. In the extracts of cells grown Eur J Blochem 57 (1975)
J. M. Lopez, B. Thorns, and H. Rehbein
429
L
0
1
2 Time ( h)
Fig. 3. Acetoin dehydrogenase and diacetylinetk~lcarbinolsynthase activities in the strains 60015 and 61411 grown in Donnellan medium + 0.1 casamino acids after induction with 20 mM acetoin. (.-Acetoin --dehydrogenase .) activity in strain 60015; (00 ) diacetylmethyldiacetylmethylcarbinol syncarbinol synthase activity in strain 60015; (W- -0)acetoin dehydrogenase activity in strain 6141 1: (O--O) thase activity in strain 61411 ~
Table 3. E.xperiment to see if uctirnted acetate urises as a product .from acetoin di.rsimilation Toluenized cells, prepared as described in Materials and Methods. were incubated with the substrates (20 mM) at 30°C for 60 inin and the formed acetyl phosphate was determined. i n addition the mixture contained: potassium phosphate buffer pH 7.0 (80 mM), NH,CI ( 5 mM), MgSOd (0.1 mM), thiamine pyrophosphate (0.025 inM), N A D (1 mM). CoA (0.1 mM), L-cystcine (1 mM), phorpliot~ansacetylase(1 U inl) Substrate
No substrate Acetoin Diacetyl Methylacetoin Acetaldehyde Acetate
FOI-mectacetyl phosphatc ninolxml~’x1i~’ < 0.01 11.3 17.9 14.0 < 0.01 < 0.01
in the presence of thiamine no stimulation of the acetoin dehydrogenase activity was obtained by the addition of thiamine pyrophosphate, indicating the presence of sufficient amounts of endogeneous thiamine pyrophosphate. Similar results were obtained for pyruvate dehydrogenase (Table 2). Activated Acetate Arises as a Product porn the Acetoin Dissimilation The postulated analogy between the pyruvate and acetoin cleavage suggests that an activated acetate, such as acetyl-CoA or acetyl phosphate is the final product from the oxidation of hydroxyethylthiamine pyrophosphate rather than the free acetate. In an attempt to clarify this problem, washed, toluenetreated cells were incubated with acetoin, methylacetoin or diacetyl in the presence of CoA and phosphotransacetylase. The acetylphosphate produced was determined (Table 3). It can be seen that acetoin or its analogs give rise to acetyiphosphate. A direct Eur J Biochem. 57 I 1975)
~
activation of acetate or acetaldehyde was excluded, since no acetylphosphate was formed in controls containing these compounds. These experiments, however, do not allow a discrimination between the formation of acetyl-CoA and acetylphosphate. Therefore a preliminary sequence of reactions is proposed :
+ (Electron acceptor),, + x Acetaldehyde + (Electron acceptor)red + Acetoin =
Thlamlnr pyrup””’’”’
’“
t
Acetyl-X
CoAS or P.
The acetaldehyde produced froin acetoin can be further metabolized as suggested by the observation that B. subtilis is able to oxidate free acetaldehyde and to utilize it to make acetate (unpublished results from Juan Lopez and Volker Wiechern). Relation between Diacetl,lmethylcarbii~ol Synthase and Acetoin Dehjdrogenase Activities In the 2,3-butanediol cycle diacetylmethylcarbinol is formed from diacetyl [l].As we have found, diacetylmethylcarbinol was produced from diacetyl only in the absence of dichlorophenolindophenol (data not shown). This indicates that the transfer of hydroxyethyl thiamine pyrophosphate to a molecule of diacetyl occurs only in the absence of a proper electron acceptor. An analogous result was obtained by Juni and Heyin [13]. They detected diacetylmethylcarbinol after incubation of diacetyl with pyruvic oxidase under anaerobic conditions. The formation of diacetylmethylcarbinol may therefore not depend on the presence of a specific diacetylmethylcarbinol synthase but rather be a unspecific side reaction which also is catalyzed by acetoin dehydrogenase. If diacetylmethylcarbinol formation and cleavage of acetoin are catalyzed by acetoin dehydrogenase, diacetylmethylcarbinol synthase activity should be defective in mutant 61411 (acetoin dehydrogenase-). This expectation was fulfilled (Fig. 3).
430
J . M . Lopez. H. Thoins, and H . Rehbein: Acetoin Dcgradation in Boc~i//i/.s .srhfi/i.\
Fraction number Fig. 4. I/~.t/rop/rohrcc / r ~ f ) ~ i ~ f / f f ~ , ~ /or1 , t i / 4~-/i ir j/ i. i i i i o h r i l ~ . ~ ~ r ~ / t / c I , ! ~ t / ( ~ - . \ c ~B. / ~ / iI c rin1 r ~ ~ .of v c ~cell-free extract (5 111%prolein) w a s applied to the coluinn ( d = 16 iiim. 1 = 60 inin). The coluinn was gradually eluted with 100 in1 buffer 1 (10 mM sodium citrate pH 6.6, 5 inM MgSO,. 1 mM EIITA. 0.1 mM thiamine p)rophosphatc), thcn Mith 5 0 in1 buffei- 2 (hull'cr 1 with 50 mM NaCl) and then with 50 in1 bull'cr 3 (bufyer 1 with 1 hl Nac'l). Fractions of 5 1111 wci-c collected and tllc activities of acetoin dehydrogenase and d i ~ ~ c e t y l i ~ l e t l ~ y l c ; ~ r b i ~ i ~ ~ l s y t h a c c were deteriiiincd. (. . . . . .) Ultt-aviolct abwrption; (0- 0 ) dincct~linctliylc~irbinol synthase activity.inl; (0---o)acetoin deli) drogenasc activit) ml: the arrows indicalc the point of bufcr change
Since diacetylmethylcarbinol is in addition thiamine-pyrophosphate-dependent (Table 2) and since both activities elute together and at the same ratio from a hydrophobic chromatography column (Fig. 4) we tentatively assume that both reactions are catalyzed by the same enzyme. The significance of thc 2,3-butanediol cyclc is an open question also because until now one reaction of the cycle, the enzymatic oxidation of acetoin to diacetyl, could not be demonstrated. Diez et a / . [14] and Gabriel et a/. [15] reported that the reduction of diacetyl to acetoin is practically irreversible. Bryn ct a/. [16] found that the enzyme diacetyl (acetoin) reductase is not able to catalyze the oxidation of acetoin to diacetyl. Other attcmpts to detect this activity with extracts of different microorganisms also where unsuccessful [17- 191. Only Sebek and Randless [20] reported that cells of Pscudomonas flziorescens produce diacetyl if grown with acetoin or 2,3-butanediol. The dichlorophenolindophcnol reduction in B. s u / ~ tilis extracts does not run at the expense of the oxidation of acetoin to diacetyl as was previously proposed [ 3 ] The . direct cleavage of acetoin explains its dissimilation without the necessity to assume its oxidation to diacetyl. Further studies concerning the detailed mechanism of the degradation of acetoin and analogous compounds can be attempted after the purification of acetoin dehydrogenase, which is in progress. This work w a s supported i n part by a grant from the Dwt.sc.lir Forsc/~~irig.s~c~ir,irc~iir.sc~lrafi. We thank Dr Thauer for instructions in handling thc gas chromatograph. Part of this work is submitted
by H . Rchbein in partial fulfilment of the requirements for the Dr rer. nat. degree in the Abteilung fiir Chcmie, Ruhr-Universitlit Bochum.
REFERENCES 1. JLini, E. & Heym, G . A. (1956) J . Buctrriol. 71, 425-432. 7 LOpez. J. & Fortnagel, P. (1972) Bkdzim. BiopIij,.~.A(,fu.279. -. 554- 560. 3. LbpeL, J., Thorns, B. & Fortnagel. P. (1973) Eur. J . Biochrrii. 40,479-483. 4. Freese, E. & Fortnagel, U . (1969) J . Bucteriol. 99, 745-756. 5. Frccse, E. & Fortnagel. P. (1967) .I. Bacterial. 94. 1957- 1969. 6. Donnellan. I. E., Nags. E. H . & Levinson, H. S. (1964) J . Buctcviol. 87, 332- 336. 7. Westerfeld, W. W. (1945) J . B i d . Chcm. 161. 495-502. 8. Lipmann, F. & Tuttlc, C. (1945) J . Biol. Chem. 159, 21-28. 9. Lowry, 0. H.. Rosebrough, M . J., Farr, A. L. & Randell, R. J. (1951) J . Biol. c'/imni. 193, 265. 10. Juni, E. & Hcym. G. A. (1956) J . Bacteiiol, 72, 746-753. 11 March. S. C., Parikh & Cuatrecasas. P. (1974) Anal. Bioc./wrn.60, 149- 152. 12. Holrcr, H. & Goedde, H. W. (1957) Biochrm. 2.320,192-208. 13. Juni, E. & Heyin, G. A. (1956) J . Biol. Clrem. 218. 365-378. 14. Diez. V . , Burgos. J . & Martin. R . (1974) Bioc~liini. Bioplrj..\. Acta. 350. 253 262. 15. Gabriel, M. A,,Jabara, H. & Al-Khalidi, U . A. S. (1971) Bioclirm. J , 124, 793- 800. 16. Bryn. K., Hctland, 0. & Stormer, F. C. (1971) Eur. J . Biocliem. 18, 116-119. 17. Seitz, E. W.. Sandive, W. E., Ellitzer. P. R . & Day, E. A. (1963) Crm. J . Microhid. 9, 431 -441. 18. Strecker, H. J. & Harary, I. (1954) J . B i d . C h m . 211,263-270. 19. Suoinalanen, H. & Linnahaline (1966) Arch. Bioc~herii.Biop/rj..c. 114, 502-513. 20. Sebek, 0. K. & Randlcs, C. I. (1952) Arch. Bioc~he,un. Biophj,.s. 40. 373 - 379. -
J . M . Lbpcz. B. Thorns. and H. Rehbein, Arbcitsgruppe Bioclieinie der Mikroorganismen, Abteilung fur Biologie der Ruhr-Universitiit Bochum. D-4630 Boclium-Querenburg. Poslracli 21 48, Federal Republic of Germany
Eur. J. Biocheni. 57 (1975)
