TIBS 17 - JUNE 1992
REVIEWS PORPI-IYRIN DERIVATIVES such as hemes and chlorophylls are important in respiratory and photosynthetic metabolic reactions. Eight molecules of the early precursor 5-aminolevulinic acid (&aminolevulinic acid, ALA) provide all the carbon and nitrogen atoms for the porphyrin skeleton of these molecules. In some bacteria and in the mitochondria of yeast, avian and mammalian cells, ALA is formed via the Shemin pathway, which involves the single-step condensation of succinyl-coenzyme A and glycine catalysed by ALA synthaseL By contrast, in the chloroplasts of plants and green algae, in cyanobacteria (e.g. Synechocystis 6803), in some eubacteria (e.g. Escherichia coli and Bacillus subtilis) and archaebacteria, ALA is formed via the C5-pathway from the five-carbon (C5) skeleton of glutamate (for reviews see Refs 2-6). Interest in the latter pathway was prompted by the discovery of an RNA cofactor 7-9, later shown to be a glutamate transfer RNA]°,1] (tRNAC~°), which was required for the biochemical transformation of glutamate to ALA. Our current understanding of this pathway is summarized in Fig. 1. The initial metabolite for the two-step Cs-pathway is the usual GIu-tRNA°u, which is converted by the action of an unusual enzyme, glutamyl-tRNA reductase (GIuTR), to glutamate-l-semialdehyde with the concomitant release of tRNAc~u. Glutamate~ 1-semialdehyde may also exist in a cyclic form, hydroxyamino-tetrahydropyranone 4 (HAT, Fig. 1). Both compounds are converted to ALA by a specific transaminase, glutamate-l-semialdehyde 2,1-aminomutase (GSA-AM;E.C. 188.8.131.52) 3,4. Thus, glutamate-l-semialdehyde is the first committed precursor of porphyrin synthesis in organisms and organelles that use this pathway. GlutRNA61ualso provides glutamate for protein biosynthesis. This review describes our current knowledge of the tRNA involvement in the Cs-pathway.
Glutamyl-transfer RNA:a precursor of heme and chlorophyll biosynthesis In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-l-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA G~uis common to the biosyntheses of both porphyrins and proteins.
tRNA=" has dual functions
Studies of ALA synthesis in extracts of barley chloroplasts, Chlamydomonas reinhardtii 7,8 and Chlorella 9 revealed that an RNA, possibly a tRNA, was required for the in vitro transformation of glutamate to ALA. Sequence analysis of the active RNA species from barley chloroplasts showed that a tRNA was indeed involved ~°. The nucleotide se-
quence (Fig. 2) demonstrated that this was a tRNA°u, containing ten modified nucleotides and the anticodon UUC, in agreement with the possible aminoacylation of this RNA with glutamate in vitro. The 3'-terminal CCA sequence of tRNA is required for aminoacylation of the molecule, so it was not unexpected that tRNAc~uwith an intact CCA end was essential for ALA formation ~°. The tRNA
PLP/PAP Glutamate-1-semial~ehyde~' (~H2 2,1-aminornutase / CH2 CHO Glu-tRNA
5-Aminolevulinic acid (ALA)
D. Jahn, E. Verkamp and D. S~II are at the Department of MolecularBiophysicsand Biochemistry,Yale University,New Haven, Connecticut, CT 06511, USA.
Figure 1 Scheme of the C5-pathway:ALA formation from Glu-tRNAG~u.Two of the proposed structures for GSA are shown4,31.
© ]992,ElsevierSciencePublishers,(]JK) 0376-5067/92/$05.00
TIBS 17 - JUNE 1992
inhibition of C. reinhardtii GIuRS by heme (>90% inhibition at 5 pM)2°and of (Barley chloroplast) the Scenedesmus enzyme by protochlorophyllide (83% inhibition at 45 pM)21 ~o. has been reported. However, heme in c A very low concentrations (_>1 pM) has ~oU been shown to disrupt protein-nucleic C-G C-G acid interactions by non-specific inhiI;-13 C-G bition of a number of enzymes, for exU-G ample, restriction endonucleases, DNA u GA o=" CU A = ~ c c c c ~ u c A I I I I I polymerases, RNA polymerases, DNA UCUG G G I I I G ligases, DNases and transcription facGAc m°~GGG~T~C D D C A A AG tor-DNA complexes 22. Thus, the physioU.GC C-G logical significance of the observed U-A C-G GIuRS inhibition is unclear. In addition, ~'°G Synechocystis GIuRS was not inhibited C A U A by high amounts (10 pM) of heme 23. We mamSs=U C therefore prefer to consider Glu-tRNA°u as a metabolite that is present in a funcGlutamyl-tRNA synthetase tional excess so that the cell can meet Figure 2 Glutamyl-tRNA synthetase (GIuRS; the needs for activated glutamate for Nucleotide sequence 1° of barley chloroplast tRNA61u. Abbreviations: ac4C, N4glutamate-tRNA ligase, E.C. 184.108.40.206) is a both protein and porphyrin synthesis. acetylcytidine; D, dihydrouridine;H', pseudomember of the well-studied class of Considering these facts, we propose the uridine; mam5s2U, 5-methylaminomethylaminoacyl-tRNA synthetases 19 and is Cs-pathway to be a two-step route to 2-thiouridine; m5C, 5-methylcytidine; T, 5responsible for the formation of Glu- ALA starting with GIu-tRNA~lu (Fig. 1). methyluridine (ribothymidine);-, standard tRNA C~u from glutamate and tRNAC~" in base pair; o, G.U pair. the presence of ATP. GIuRS enzymes Glutamyl-tRNA reductase from several organisms possessing the GIuTR catalyses the NADPH-depennature was also illustrated by the com- Cs-pathway have been purified (see Ref. dent reduction of GIu-tRNAGlu to glutaplementation of E. coil tRNAG~" with 14 for recent review). GIuRS from mate-l-semialdehyde or a similar interChlamydomonas enzyme fractions in in chloroplasts and Gram-positive eubac- mediate with the release of free t R N A Glu, vitro ALA formation u. More recently, teria can also form Glu-tRNA°°, an inter- which can then be recharged with gluthe RNA sequence of the tRNAeL"active mediate in the formation of GIn-tRNAc~n tamate. Investigations of this reaction in ALA formation in C. reinhardtii in these organisms. The same GluRS is have been hampered by the scarcity of chloroplasts was determined ~2. responsible for charging tRNA°u for purified enzyme, which is apparently A sequence comparison of tRNAc~" both protein and heine biosynthesis. present in low abundance in the cell. GIuRS has been considered as the Our current knowledge of GluTR from a genes from chloroplasts of spinach, broad bean, tobacco, wheat, pea, liver- first enzyme of the Cs-pathway. How- number of species is summarized in wort, Euglena gracilis, Chlamydomonas, ever, a definition of a pathway is that its Table I. The number and molecular from the cyanelle of Cyanophora para- enzymes are regulated in an inter- masses of the enzymes from different doxa, and several prokaryotic organisms dependent fashion or that their activity sources vary and much additional work possessing the C5-pathway has revealed may be subject to feedback inhibition is needed to define the similarities some features unique to the tRNAclu by downstream metabolites. The cur- between these enzymes. In Chlamydoinvolved in porphyrin biosynthesis 2J3,14. rently available published data do n o t monas 24, a monomer of 130 kDa was With the exception of E. coil and B. sub- provide convincing support that GIuRS purified, while two E. coil proteins had tills tRNAGlu,all possess an As3"U61base is regulated either by chlorophyll pre- GIuTR activity with molecular masses pair at the same l~osit.ion in which most cursors or in concert with the other of 45kDa (GluTR45) and 85kDa elongator tRNAs have a highly c o n - enzymes of ALA biosynthesis. In vitro (GIuTR85)25. By contrast, the GIuTR of Synechocystis 6803 is easily purified; the enzyme contains eight identical 39 kDa Table I. The purified enzymes and characterized genes of glutamyl-tRNA reductase subunits 26. Organism Subunit molecular Quaternary Enzymepurified Genecloned Reference Genes encoding GluTR have been mass (Da) structure isolated from a number of eubacteria by complementation of the hemA muC. reinhardtii 130 000 (~ + 24 tation of E. coil (Table I). That hemA Synechocystis 39 000 (~8 + 26 genes encode GluTR, or a structural 47 525* ? + 29 component of this enzyme, was first E. coil 85 000 (~ + 25 demonstrated for a cloned Chlorobium 46 254* (z + + 25, 46-48 gene. This gene showed significant S. typhimurium 46 080* ? + 49 amino acid sequence homology27 to B. subtilis 51800* ? + 30 other cloned hemA genes and compC. vibrioforme 46 174" ? + 27 lemented the E. coil hemA mutatiOn, but conferred Chlorobium tRNA specificity *Calculated from the gene sequence. o n the GluTR activity in the transtRNA Glu
served G.C pair. It is unknown whether these nucleotides play a role in the RNA recognition by GIuTR. Initially it was suggested that the RNA involved in ALA formation may be a special tRNA species ~°. However, sequence determination of all glutamate-acceptor RNAs of barley chloroplasts indicated only one tRNAc~" species ~s, in agreement with the existence of only one gene encoding tRNATM in the barley chloroplast genome~°JE Biochemical evidence that the same tRNATM supports ALA formation in addition to protein biosynthesis was obtained for GIu-tRNA~lu from Synechocystis ~7,1a and C. reinhardtii ~4. Thus, the usual tRNA°~ is the substrate for GIuTR.
TIBS 1 7 - JUNE 1 9 9 2
formed E. coli strain 2~. Direct proof that the E. coli hemA gene encodes GluTR45 was provided by the heterologous expression of the E. coli HemA protein in the yeast Saccharomyces cerevisiae 29, which does not possess the C~-pathwayL The partially purified protein overexpressed from yeast was shown to possess GluTR activity by the in vitro conversion of E. coli GIu-tRNATM to GSA; gel filtration studies showed it to have a molecular mass of 45 kDa. An E. coil hemA strain, deficient in GluTR45, has GiuTR85 activity and grows anaerobically on glucose-containing medium ~9. Therefore, GluTR85 is not affected by the hemA mutation and may serve some anaerobic ALA requirement of the cell. The relationship of the purified GluTR from Synechocystis ~ to the cloned hemA homoiog from this organism 29 needs to be examined. The amino-terminal peptide sequence deduced from the Synechocystis gene structure and highly conserved in the hemA-derived sequences of five organisms (Fig. 3) is not contained in the amino acid sequence determined from the purified Synechocystis protein. This fact, along with the rather different GluTR structure, suggests that Synechocystis may, like E. coli ~5, have two GIuTR activities, although only one activity was detected during purification2~. The deduced amino acid sequences of prokaryotic GluTR genes display up to 60% similarity with some highly conserved regions (Fig. 3). Domains such as the pyridine nucleotide-binding domain may show evolutionary conservation, while other variable amino acid sequences might confer specificity for recognition of tRNA or cellular regulatory components. There is a conserved stretch of 23 amino acids with 52% identity between positions 99-121. In B. subtilis an amino acid substitution (Cysl05Tyr) within this region results in a HemA- phenotype ~°. NADPH is the only required cofactor for the activity of the purified GIuTR enzymes 24-26. High concentrations of NADH substituted for NADPH when tested with the Synechocystis enzyme2~, while ATP and GTP did not influence GIuTR activity2~. The structure of glutamate-l-semialdehyde, the reaction product from GluTR, also needs further study. While initially reported to be a linear molecule 3~, a more stable cyclic form, HAT, has recently been proposed 4 (Fig. 1). What is the nature of the macromolecular substrate, tRNAG~u?All GluTR
I E.coli S.typh B.subt Synech C.vibr Consen E.coli S.typh B.subt Synech C.vibr Consen
~VS~R ~ I E ~
~IAV~fLSH R ~ v E ~ IIS
VTFSPDTLDQ A L D ~ L A Q P M VSFQPNELAE AMVQ~KEEKS ALT~RSYPH R IALSEVQNKE FVT VSSGL V-F .... L-- AL--~ . . .
VQGGVV~T~LSVE ILENII~I~AVVD IEEVTV
EQDNLQEALI QLHTGRYYIK A V V T DTEKGVVEIT MPEVNCDYLK . . . . . . . . . . . . . .
81 EEDLRKSLYW H Q D N D A V ~ EDDLRNSLYW H Q D N D A V ~ KEELSPFLTF Y E S D A A V ~
MF~ASGLI~L ~ FBCa~ACGLDmM ~ D B I T ~ 3 ~
---L---L. . . . . . . AV-~ -~A-GIJI~L~ ( : ~
GHLNASALRR EKTIGTIFNE YKGVGRLLDR ~KDAYRIAAE VGTAGILLTR ~---F---Q- ....... L-R
E.coli S.typh B.subt Synech C.vibr Consen E.coli S.typh B,subt Synech C.vibr Consen
RWLCDYHNLN KFLADWFQLS QFLSETGNIP DYIISYKDAR L-DY .... 160
MFQKSFSVAK MFQKSFSVAK LFKQAVTVGK LFKQAITAGR LCHSPFSVAK LF ..... V-K
RVRTIDIGA RVRT RTHA RVRT KVKT RV-T
DIGA DIGS DIGT KLME DIG240
T~ARQIFE~ ~TVTVLL~ T~RQIFE~ ~TVTVLL~
GETI.ELVAR HLREHKVQKM IIANRTRERA GETI.ELVAR HLREHKVQKM IIANRTRERA . Q ~ D E V G A TVINRTYLKA . K ~ D R F S G GKMA.CLLVK HLLAKGATDI TIVNRSQRRS . Q ~ Q F P Q
~ I S ~ V
~o~i~s~ | . ~ v ~
~0sw~s= ~ s ~
~ V - ~ -
-~A- - IF- 2~3
G ..... L . . . . L ........ - I - N R - - - R A
241 RLR...ER~ RL...QD~ ..SALA~ MFTAI HLHEF...~
IISSTASPLP IISSTASPLP L ISSTGASEF VFTSTGATEP IITAVSTKEY
--~:~ ~ - S T . . . . .
IIGKGMVERA IIGKGMVERA VVSKEMMENA ILNCENLTGC ILNAAEMQQS
~. . . . . . . . . . . .
LKS~RNQPML LK~RNQPML NKLRKGRPLF VI~S..LM MA~RLKPVI
L~IA~DV L IA~V ~IA L ~ I S ~ I LG
EVISLSDIDA EARSLNQLE. AQLTLCPLTD
EPEVGKLANA YLYS .... EPEVGKLANA Y L Y S ~ S
320 S IIS L A I I ~ Q
AADVHAMEQV RAF E W DPEVGALQNM F L K D ? ~ K H IID~L_~_RLER
E.coli S.typh B.subt Synech C.vibr Consen
321 AAAVEAETIV AAAVEAETIV ETAEKVELLI QMARQAEALL AELPKVKSI I A E --
4OO AQETSEFMA . . . . . . . W L R A QSASETIREY ~SQAEQVRDE LTAKALAAL. .EQGGDAQAI MQDLAWKLTN EQEASEFMA. .WLRA QGASETIREY ~SQSEQIRDE LTTKALSALQ Q..GGDAQ~I LQDLAWKLTN EETIVEFKQ. .WMNT LGVVPVISAL ~EKALAIQSE TMDSIERKL. PHLSTREKKL LNKHTKSIIN EEEIAAFDL. .WWRS LETVPTISSL RSKVEDIREQ ELEKALSRLG SEFAEKHQEV IEALTRGIVN DEELIASASG STPSRYVRPS LTCNPSSSKS ~RKNSSVPPQ GERRGVEAHG T .... PDRQD PEKNPASSYQ E-E---F . . . . . . . . . W . . . . . . . . . I---iR ..... I . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . N
E.coli S.typh B.subt Synech C.vibr Consen
401 RLIHAPTKSL RLIHAPTKSL QMLRDPILKV KILHEPMVQL DAQGSGRYRR P
472 Q.Q...AARD GDNERLNILR DSLGL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q.Q...AARD GDDERLNILR DSLGLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K,..ELAADA DSEEKLALFM QIFDIEEAAG GRQMMKTVES SQKVHSFKKA ESKAGFSPLV SE KAQQDIEARK QCLRSLKMLF D.LEVEEQFG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QHPQQSQPRQ EHLRS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AR . . . . . . L--L- --L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rgum3 Alignment of amino acid sequences of the known Glu-tRNA reductase structures deduced from the cloned hemA genes of B. subti/is 3°, C. vibrioforme 27, E. co/i 25'46~8, S. typhimurium 49 and Synechocystis 29. The consensus sequence lists amino acids in positions that contain similar amino acids in at least four out of five HemA proteins. Tinted residues are identical in all known sequences.
enzymes tested so far can discriminate between different tRNA species. For example, the barley GIuTR can utilize charged chloroplast tRNAGlu as a substrate, but not E. coil or barley cytoplasmic Glu-tRNA~" (Ref. 32). By contrast to all the chloroplast tRNATM species, the E. coil tRNA does not possess the Ass'U6, base pair, although many other bases are conserved between these tRNAs ~4. The E. coli tRNA is also a good substrate for barley chloroplast GIuRS~5. This base pair could therefore be important for tRNA~'u recognition by GIuTR from higher plants. By contrast to barley GluTR, the Chlamydomonas enzyme can recognize E. coli tRNAclu in addition to its homologous t R N A 8'33. Transfer RNA specificity has also been demonstrated for GluTR enzymes from Chlorobium and Chlorella 28,34. It appears that GIuTR can discriminate between different tRNA molecules even if they are aminoacylated with glutamate. Thus, any recognition of the amino acid by GIuTR must be secondary to that of the tRNA. Two different mechanisms for the GluTR-catalysed reduction have been suggested 6'24. One mechanism 24 is formally analogous to the back reaction of
glyceraldehyde-3-phosphate dehydrogenase (E.C. 220.127.116.11), where the phosphorylation-activated carboxyl group of 3-phosphoglycerate is reduced to its aldehyde in the presence of NADH via an acyl-enzyme intermediate. In the case of GluTR, the glutamate is activated by ligation to tRNA. The other proposal suggests the presence of a complex of GIuRS, GluTR, Glu-tRNAc'u and GTP6, which acts as the 'substrate' for another enzyme initiating the reduction. However, the generation of glutamate-1semialdehyde from Glu-tRNATM by all purified GluTR enzymes in the presence of NADPH alone 24-26, as well as the in vitro transformation of glutamate to ALA35 by three purified Chlamydomonas enzymes (GluRS, GIuTR, GSA-AM) with only their substrates and cofactors present (tRNAc~°, ATP, NADPH, pyridoxal 5'phosphate), renders the latter mechanism unlikely. Heine inhibition of pure GIuTR preparations has also been studied to gain more insight into the regulation of the Cs-pathway. The purified enzymes from E. coli were found to be insensitive2s to this tetrapyrrole, but the purified Synechocystis enzyme was inhibited by heme (50% at 5-10 I~M)26. Similar con-
TIBS 17 - JUNE 1992 Biochem. Biophys. 239, 87-93 second tRNA-dependent pathway for Sch6n, A. et al. (1986) Nature 322, 281-284 ALA synthesis could exist. Genetic and 10 11 Huang, D. D. and Wang, W-Y. (1986) J. Biol. biochemical studies in E. coil and the Chem. 261, 13451-13455 further characterization of GIuTR ac- 12 O'Neill, G. P. et al. (1990) Nucleic Acids Res. 18, 5893 tivities should provide an answer. 13 Sprinzl, M., Dank, N., Nock, S. and Sch6n, A. Another unanswered question con(1991) Compilation of tRNA and tRNA Gene cerns the flow of Glu-tRNABl" into proSequences (1991 edn), Laboratorium~l~r Biochemie, Universit~t Bayreuth, Germany tein or porphyrin synthesis. Although there is only one gene for this tRNA in 14 Jahn, D., O'Neill, G. P., Verkamp, E. and S611,D. (1992) Plant Physiol. Biochem. 30, 245-253 barley chloroplasts m6, different mature 15 Sch6n, A., Kannangara, C. G., Gough, S. and S611, D. (1988) Nature 331, 187-190 tRNA molecules with varying degrees of Glutamate-l-semialdehyde2,1-aminomutase nucleotide modification do exist TM. 16 Berry-Lowe, S. (1987) Carlsberg Res. Commun. 52,197-210 This enzyme catalyses the GSA~ALA These differences may be important for 17 Schneegurt, M. A., Rieble, S. and Beale, S. I. transamination in the presence of pyri- the interaction of tRNAc~uwith GluTR or (1988) Plant Physiol. 88, 1358-1366 doxal 5'-phosphate (PLP) or pyridox- components of the protein-synthesizing 18 O'Neill, G. P. and S611,D. (1990) J. Bacteriol. 172, 6363-6371 amine 5'-phosphate (PAP). It has been system. Alternatively, the flow of GIu19 Schimmel, P. R. and S611,D. (1979) Annu. Rev. purified from bacteria and chloroplasts tRNAc~" into the different pathways may Biochem. 48, 601-648 of higher plants, and the genes en- be determined by the relative concen- 20 Chang, T. E., Wegmann, B. and Wang, W. Y. (1990) Plant Physiol. 93, 1641-1649 coding this protein have been charac- trations of elongation factor Tu (EF-Tu) D. et al. (1989) Bot. Acta 102, terized 3s,37-42. Most of the enzymes have and GluTR, two proteins that compete 21 D6rnemann, 112-115 a dimeric structure with subunit mol- for binding GIu-tRNAG~u. 22 Halle, D. J. et al. (1990) J. Biol. Chem. 265, 12768-12789 The ability of GIuTR to discriminate ecular masses of 44--46 kDa. A nuclear gene (gsa) encodes this enzyme in between structurally very similar tRNA 23 Rieble, S. and Beale, S. I. (1991) Arch. Biochem. Biophys. 289, 289-297 higher plants 37. The corresponding gene molecules adds another interesting di- 24 Chen, M. W., Jahn, D., O'Neill, G. P. and S611,D. in E. coli, B. subtilis and S. typhimurium mension. The comparison of the identity (1990) J. Biol. Chem. 265, 4058-4063 is hemL 4°-42. E. coli and S. typhimurium elements in tRNAclu for GIuTR recog- 25 Jahn, D., Michelsen, U. and S611,D. (1991) J. Biol. Chem. 266, 2542-2548 strains carrying hemL mutations as well nition with those for aminoacylation 45 26 Rieble, S. and Beale, S. I. (1991) J. Biol. Chem. as a B. subtilis strain harboring a hemL by glutamyl-tRNA synthetase will give 266, 9740-9745 deletion-substitution display a leaky further insight into the manifold versa- 27 Majumdar, D., Avissar, Y. J., Wyche, J. H. and Beale, S. I. (1991) Arch. Microbiol. 156, 281-289 phenotype regarding ALA auxotrophy4~42. tility of tRNA for protein recognition. 28 Avissar, Y. J. and Beale, S. I. (1990) As the first reported reduction proInactivation of the gene resulted in a J. Bacteriol. 172, 1656-1659 slow-growing strain; addition of ALA cess involving tRNA, detailed analysis 29 Verkamp, E. et al. (1992) J. Biol. Chem. 267, 8275-8280 restored the wild-type phenotype 42. of the GIuTR reaction may reveal novel 30 Petricek, M., Rutberg, L., Schr6der, I. and These observations led to speculations biochemical mechanisms. This reaction Hederstedt, L. (1990) J. Bacteriol. 172, that other aminotransferases with low may also presage the discovery of other 2250-2258 substrate specificity may substitute for processes in which tRNA serves to acti- 31 Houen, G., Gough, S. P. and Kannangara, C. G. (1983) Carlsberg Res. Commun. 48, 567-572 GSA-AM. The gene-derived amino acid vate acyl groups for still unknown func32 Peterson, D., Sch6n, A. and S611,D. (1988) sequences of GSA-AM enzymes show tions of cellular metabolism. Plant Mol. Biol. 11, 293-299 significant homology and contain a 33 O'Neill, G.P. et al. (1988) J. Bacteriol. 170, 3810-3816 highly conserved sequence predicted Acknowledgements 34 Weinstein, J. D., Mayer, S. M. and Beale, S. I. We thank A. Lloyd, G. O'Neill and to be involved in the binding of the (1986) Plant Physiol. 82, 1096-1101 cofactor39. Alternatively, slow chemical M. J. Rogers for discussions. Work in 35 Jahn, D., Chen, M. W. and $611, D. (1991) J. Biol. Chem. 266, 161-167 conversion of glutamate-l-semialdehyde the authors' laboratory was supported 36 Javor, G. T. and Febre, E. F. (1992) J. BacterioL to ALA43 may explain the leakiness of by grants from DOE and NIH. 174, 1072-1075 hemL mutants. 37 Grimm, B. (1990) Proc. Natl Acad. Sci. USA 87,
centrations (5 gM) of heme, but not of protoporphyrinogen IX, inhibited GluTR activity in E. coli extracts 36. This apparent contradiction may be resolved if additional factors are needed to mediate heme inhibition or if heme is converted to an inhibitory metabolite by E. coli extracts. For example, 1-thioglycerol strongly stimulated GIuTR synthesis in aerobically grown E. coli 36.
Outlook The formation of ALA is known to be a key regulatory step in chlorophyll and heme biosynthesis in many organisms as both feedback inhibition and light regulation have been shown 2-4. However, levels of tRNAc~uand Glu-tRNA°" in barley chloroplasts and Synechocystis 16,18 and mRNA levels of barley GSA-AM37are not affected by changes in illumination. The availability of purified enzymes and of cloned genes will permit detailed analyses of the regulation of enzyme activity and of gene expression. Why are there two GluTR activities in E. coli? Since the hemL mutations are leaky4e-42 and since there may be two ALA pools in cucumber chloroplasts 44, a
1 May, B. K. et al. (1986) Curr. Top. Cell. Regul. 28, 233-262 2 O'Neill, G., Jahn, D. and S611,D. (1991) in Subcellular Biochemistry (Vol. 17): Plant Genetic Engineering(Biswas, B. B. and Harris, J. R., eds), pp. 235-264, Plenum 3 Beale, S. I. and Weinstein, J. D. (1990) in Biosynthesis of Heine and Chlorophylls (Dailey, H. A., ed.), pp. 287-391, McGraw-Hill 4 Jordan, P. M. (1990)in Biosynthesis of Heine and Chlorophylls (Dailey, H. A., ed.), pp. 55-121, McGraw-Hill 5 O'Neill, G. P. and S611,D. (1990) Biofactors 2, 227-235 6 Kannangara, C. G. et al. (1988) Trends Biochem. Sci. 13, 139-143 7 Kannangara, C. G., Gough, S. P., Oliver, R. P. and Rasmussen, S. K. (1984) Carlsberg Res. Commun. 49, 417-437 8 Huang, D-D., Wang, W-Y., Gough, S. P. and Kannangara, C. G. (1984) Science 225, 1482-1484 9 Weinstein, J. D. and Beale, S. I. (1985) Arch.
4169-4173 38 Pugh, C. E., Harwood, J. L. and John, R. A. (1992) J. Biol. Chem. 267, 1584-1588 39 Grimm, B., Bull, A. and Breu, V. (1991) Mol. Gen. Genet. 225, 1-10 40 Ilag, L. L., Jahn, D., Eggertsson, G. and $611,D. (1991) J. Bacteriol. 173, 3408-3413 41 Elliott, T., Avissar, Y. J., Rhie, G-E. and Beale, S. I. (1990) J. Bacteriol. 172, 7071-7084 42 Hansson, M., Rutberg, L., Schr6der, I. and Hederstedt, L. (1991) J. Bacteriol. 173, 2590-2599 43 Hoober, J. K. et al. (1988) Carlsberg Res. Commun. 53, 11-25 44 Huang, L. and Castelfranco, P. A. (1990) Plant Physiol. 92,172-178 45 S611,D. (1990) Experientia 46, 1089-1096 46 Verkamp, E. and Chelm, B. K. (1989) J. Bacteriol. 171, 4728-4735 47 Drolet, M. et al. (1989) Mol. Gen. Genet. 216, 347-352 48 Li, J. M., Russell, C. S. and Cosloy, S. D. (1989) Gene 82,209-217 49 Elliott, T. (1989) J. Bacteriol. 171, 3948-3960