Proc. Natl. Acad. Sci. USAVol. 83, pp. 867-871, February 1986Biochemistry

Evolution in biosynthetic pathways: Two enzymes catalyzingconsecutive steps in methionine biosynthesis originate froma common ancestor and possess a similar regulatory region

(homology/,8-cystathionase/cystathionine y-synthase/sequence/operator)

JAMILA BELFAIZA*, CLAUDE PARSOT*, ANNIE MARTELt, CLAIRE BOUTHIER DE LA TOURt,DANIELLE MARGARITA*, GEORGES N. COHEN*, AND ISABELLE SAINT-GIRONS*

*Unlitt de Biochimie Cellulaire, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris C6dex 15, France; and tLaboratoire de Bioorganique etBiotechnologies, Ecole Nationale Supdrieure de Chimie de Paris, 11, rue Pierre et Marie Curie, 75231 Paris Cddex 05, France

Communicated by Edmond H. Fischer, September 30, 1985

ABSTRACT The metC gene of Escherichia coli K-12 wascloned and the nucleotide sequence of the metC gene and itsflanking regions was determined. The translation initiationcodon was identified by sequencing the NH2-terminal part of13-cystathionase, the MetC gene product. The meIC gene (1185nucleotides) encodes a protein having 395 amino acid residues.The 5' noncoding region was found to contain a "Met box"homologous to sequences suggestive of operator structuresupstream from other methionine genes that are controlled bythe product of the pleiotropic regulatory metJ gene. Thededuced amino acid sequence of (3-cystathionase showed ex-tensive homology with that of the MetB protein (cystathioniney-synthase) that catalyzes the preceding step in methioninebiosynthesis. The homology strongly suggests that the struc-tural genes for the MetB and MetC proteins evolved from acommon ancestral gene.

It has been established that the expression of the methioninegenes in Escherichia coli is repressed in a parallel butnoncoordinate manner by methionine (1). The regulation ismediated through the product of the metJ gene (2-5). Geneticexperiments (6, 7) as well as recent in vitro studies (8) indicatethat S-adenosylmethionine interacts with the aporepressorspecified by the MetJ gene product.

Cystathionine y-synthase and P3-cystathionase encoded bythe metB and metC genes located at 88 and 65 minutes on theE. coli chromosome, respectively (9), mediate consecutivereactions in the methionine pathway (1). Their physicochemi-cal properties are similar since these two enzymes arecopurified in many types of chromatographic systems (un-published results) and purification of cystathionase fromSalmonella was reported as a by-product of cystathioniney-synthase purification (10). In addition, cystathionine y-synthase has a broad specificity with respect to substratestructure (1) and can catalyze the reaction of H2S withO-succinylhomoserine to give homocysteine directly, thusbypassing the cystathionine intermediate (1). However, thislast reaction cannot provide a major alternative pathwaysince metC mutants lacking A3-cystathionase, although slight-ly leaky (11), have indeed been selected as methionineauxotrophs.

In this paper, we report the cloning and the DNA sequencedetermination of the metC gene and its flanking regions. The5' region of the metC gene was compared to that of the othermethionine genes that share the same regulatory mechanism.In addition, we found the MetB (12) and MetC proteinsequences to be highly homologous, indicating a commonorigin in evolution.

MATERIALS AND METHODSChemicals. L-Cystathionine was from Sigma; blue Sepha-

rose CL-B6 and octyl-Sepharose 4B were from Pharmacia;DE-52 cellulose was from Whatman. All other chemicalsused were of analytical reagent grade. dATP[a-35S] or [y-32P]dATP and the M13 sequencing kit from Amersham wereused. Restriction enzymes were from Boehringer Mannheimor New England Biolabs.

Bacterial Strains and Growth Media. E. coli K-12 strainsused throughout this work are as follows. Strain GUC41,exbB, AmetC, thr, leu, tonA (13) was kindly provided byRonald Greene. Strain HfrH and strain AT2446 Hfr, thi-J,metC69, relAl, spoTI were obtained from the Institut Pasteurcollection. Gif 881-L is HfrH metJ (14). Strain CSR603 (15),thr-1, leuB6, proA2, phr-J, ara, recAl, argE3, uvrA6, lacYI,galK2, xyL5, met-i, rpsL31, tsr33, supE was used for maxicellproduction. Media employed are described by Miller (16).

Purification and Assay of Cystathionase. Cystathionase waspurified from 50 g (wet weight) of Gif 881-L strain containingthe pIP28 plasmid (see Results), overproducing cystathio-nase. Bacteria were cultivated in LB medium supplementedwith ampicillin (50 ,g/ml) and harvested by centrifugation.The cells were suspended in 10 mM sodium phosphate buffer(pH 7) containing 10 mM pyridoxal 5'-phosphate and 1 mMdithioerythritol (buffer A) and broken by sonication. Thesuspension was centrifuged at 20,000 x g for 25 min. Theenzyme was purified by a modification of the procedure ofDwivedi et al. (17), in which the second step, chromatographyon Bio-Gel hydroxyapatite, was omitted. The DE-52 cellu-lose chromatography was immediately followed by blueSepharose CL-6B chromatography. The fractions obtainedfrom the chromatography on a Cibacron blue SepharoseCL-6B column that had specific activities >30 units/mg werepooled, adjusted to 1 M (NH4)2SO4, and loaded on a columnof octyl-Sepharose 4B preequilibrated with buffer A contain-ing 1 M (NH4)2SO4. After washing with the same buffer theenzyme was eluted with a double linear gradient of decreas-ing (NH4)2SO4 concentration (down to 0%, wt/vol) andincreasing ethylene glycol concentration (up to 75%, wt/vol)in buffer A. The purified enzyme showed a single band ofprotein by electrophoresis under native and denaturing con-ditions. The molecular weight of the subunit was found to be42,000 ± 1000.

Cystathionase activity was measured by a coupled assaysystem, as described by Guggenheim (10). One unit ofenzyme activity represents 1 ,umol of product formed per minat 370C.

Analytical Procedures. Throughout the purification, theprotein concentration was estimated by the method of

Abbreviation: kb, kilobase(s).

867

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868 Biochemistry: Belfaiza et al.

Bradford (18). With the purified enzymatic fractions, theprotein concentration was determined according to Lowry etal. (19).NH2-Terminal Sequence Determination. The NH2-terminal

sequence of cystathionase was determined with an automaticsequencer (SOCOSI PS1000 modified) from 26 nmol (in termsof monomer) of protein extensively dialyzed against distilledwater. A high-performance liquid chromatography system(Waters) was used to identify the phenylthiohydantoin aminoacid derivatives that were eluted with a methanol gradientfrom a Merck Lichrospher 60 CH8 super column.

Nucleotide Sequence Determination. The nucleotide se-quences were determined primarily by the dideoxy method(20, 21) using dATP[a-35S] and for two fragments afterlabeling at the 3' OH end with [y32P]dNTP by the chemicalmethod of Maxam and Gilbert (22).

RESULTS AND DISCUSSION

Cloning of the meiC Gene and Overproduction of Cystathio-nase by the pIP29 Recombinant Plasmid. Clarke and Carbon(23) have constructed an E. coli clone bank that carries hybridplasmid of ColEl and randomly sheared fragments of total E.coli DNA. In their gene-protein index of E. coli K-12,Neidhardt et al. have assigned the metC gene to the pLC4-14recombinant plasmid (24).We isolated pLC4-14 (kindly provided by Mathias Spring-

er) and verified that it conferred the Met' phenotype to ametC strain (AT2446). We then subcloned a BamHI-BamHIfragment from pLC4-14 into the BamHI site of pBR322 andselected for an AmpR and Met' phenotype. A 15-kilobase(kb)-long recombinant plasmid called pIP28 (see Fig. 1) wasobtained, which contained only one BamHI site indicative ofa cloning artifact. pIP28 was subsequently shortened by apartial HindIII restriction cleavage, with maintenance ofselection for the Met' character. The 4.5-kb-long hybridplasmid obtained was designated pIP29. We verified thatpIP29 conferred the Met+ phenotype to a strain with adeletion of the metC gene (GUC41). The only proteinexpressed by the bacterial insert of pIP29 in maxicells (Fig.2) was a protein of 42,000 molecular weight that comigrateswith the purified cystathionase (data not shown).The overproduction of cystathionase was investigated

further. The Gif 881-L strain (14) carrying a mutation in themetJ regulatory gene was already overproducing the methio-nine biosynthetic enzymes, in particular, cystathionase,12-fold compared to the wild type (0.017 unit/mg for the HfrHstrain). The overproduction reported with pLC4-14 in anoth-er metJ strain was 100-fold (17). Therefore, we transformedGif 881-L (metJ) by pIP28 and pIP29 and obtained a 240-foldoverproduction of cystathionase as compared to wild type.

pLC4-1 4

B B P

I I I

B

P HH B B P HB

A

P HH 'P HB,,- ,

I II I0 nIPPAi

1 kb

HH P H

metC

1 2 3

_w 115

67W FIG. 2. Autoradiogram of [35S]meth-ionine-labeled proteins from maxicellpreparations run on a 10% NaDodSO4/

37 polyacrylamide gel. Lane 1, CSR603(pBR322); lane 2, CSR603 (pIP29); lane3, iodinated markers. The molecular

O 22 masses (kDa) of the marker proteins arelow 14 shown. The radioactive band corre-

sponding to cystathionase is indicated byan arrow.

The hyperproducing strain Gif 881-L that contained theconstructed pIP28 plasmid was used to purify P-cystathio-nase. The purification steps are summarized in Table 1.

Determination of the Nucleotide Sequence of the metC Gene.The nucleotide sequence of the insert carried by the pIP29plasmid has been determined according to the strategypresented in Fig. 3. Each region was sequenced at least fourtimes and 89% of the sequence was obtained on both strands.Analysis of the sequence presented in Fig. 4 shows that anopen reading frame begins at position 316 and ends at position1686. To determine the beginning of the metC gene, wepurified cystathionase and determined the first 10 amino acidresidues of its NH2-terminal end. The results given byautomated sequencing (Ala-Asp-Lys-Lys-Leu-Asp-Thr-Gln-Leu-Val) allow the unambiguous choice of the ATG inposition 499 as initiator codon of the metC gene. It may benoted that the NH2-terminal methionine residue is absentfrom the purified protein. The gene is thus 1185 nucleotideslong and encodes a single polypeptide chain of 395 residueswith a molecular weight of 43,032. This is in agreement withthe monomer molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis (see purification in Ma-terials and Methods). The amino acid composition of theMetC protein deduced from the nucleotide sequence (Table2) is in good agreement with that determined experimentallyfrom purified MetC protein (17).The data presented here show that the coding region of the

metC gene contains a HindIII and an EcoRI restriction site.In addition, deletion of the HindIII-HindIII fragment frompIP28, containing the promoter and the first five codons ofthe metC gene, gave rise to a recombinant plasmid that didnot confer a Met' phenotype to a metC strain. Our resultstherefore disagree with those reported independently by

pBR322

FIG. 1. Construction of plasmids pIP28 and pIP29containing the metC gene. Cloning of the BamHIfragment of pLC4-14 (23) into the BamHI site ofpBR322 (25) gives rise to the pIP28 plasmid, whichcontains only one BamHI site as a result of a deletion(A) around one of the cloning sites. However, restric-tion analysis of pLC4-14, nucleotide sequence of thevector-insert junction in pIP29, and expression of theMetC gene product encoded by pLC4-14, pIP28, andpIP29 indicate that the deletion does not affect themetC structural gene. Plasmid pIP29 derives frompIP28 by a partial HindIII digestion followed by liga-tion. All plasmids are presented in a linear form onwhich the relevant restriction sites are indicated: P,Pvu II; B, BamHI; H, HindIII. The thick line repre-sents pBR322 vector DNA.

Proc. Natl. Acad Sci. USA 83 (1986)

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Table 1. Purification of 3-cystathionaseCystathionase Total Specific

Volume, activity, protein, activity, Purification Yield,Fraction ml units mg units, mg factor %

Crude extract 1100 6589 8030 0.82DE-52 cellulose 33 2797 420 6 8 42Cibacron blue

Sepharose CL-6B(and concentration) 50.7 2080 42 49 60 32

Octyl-Sepharose 4B 18.8 1170 4.7 248 302 18

Michaeli and Ron (27), who located the metC gene betweentwo HindIII sites and between two EcoRI sites.

Analysis of the 5' and 3' Flanking Regions of the metC Gene.We have found by construction of a gene fusion betweenmetC and the lacZ gene of the pMC1403 plasmid (28) thatthere is a promoter activity associated with the insert ofpIP29consistent with the presence of the metC promoter (unpub-lished data). No evidence for attenuation control (for areview, see ref. 29) has been found in any of the methionineregulatory regions sequenced so far. Indeed, the nucleotidesequence of the 499-base-pair DNA fragment located up-stream from the metC structural gene (Fig. 4) does not revealany structural features characteristic of regulation by atten-uation.

All ofthe methionine genes with the exception ofmetH andmetG are subject to repression by methionine (1) and therepression is mediated through the MetJ gene product, acommon aporepressor. It is therefore reasonable to assumethat the repressor binding sites should be similar for all of thegenes regulated via repression by methionine. Indeed, DNAsequences with a 2-fold symmetry in the regions upstreamfrom the meWf and metBL transcriptional units were suggest-ed as possible binding sites for the MetJ gene product andwere called "Met boxes" (12, 30). Michaeli et al. (31)compared then the 5' region of the metA gene to those ofmetF and metB and found an extensive homology, althoughno common axis of symmetry was found. In the DNAupstream from the coding region of the metC gene, a Met boxhas also been detected (Fig. 5). Bearing in mind that the fourMet boxes cited above are homologous, it is worthwhile tonote that additional base matches are present between theMet boxes of metC and metB (Fig. 5A) on one hand andbetween those of metA and metF (31) on the other hand.

Moreover, the present comparison between the four Metboxes (for metC, -B, -A, -F) revealed the existence of arepetitive unit (R) eight nucleotides long. As a consequence,

I500

multiple alignments could be drawn by sliding each sequencewith an eight-nucleotide periodicity. In the alignment pre-sented in Fig. 5B, of the 128 positions considered, 89 matchesand 21 transitions are found when the repetitive units arecompared to their consensus sequence R. This consensussequence is a perfect palindrome, AGACGTCT, which ispresent under an altered form, two to five times in the Metboxes (Fig. 5B). It is possible that the differences between theMet boxes are related to the different extents of repressionelicited by the MetJ gene product, as already mentioned byMichaeli et al. (31): the ratios of derepressed versus fullyrepressed levels were found to be 6-12 for metC (ref. 1; thispaper), 40 for metB, 100 for metF, and 300 for metA (1).We must recall that the metB and metJ genes are tran-

scribed divergently and could share the same Met box (32).In addition, after elucidation of the metK gene sequencecoding for S-adenosylmethionine synthase, an enzyme thatutilizes methionine as a substrate, Markham et al. (33)assigned a Met box to its 5' flanking region. However, theauthors introduced a gap 13 nucleotides long in the 26-nucleotide-long Met boxes of the metB and metF genes inorder to maximize the overlap and to keep the same axis ofsymmetry. We have thus not considered the metK gene in ourdiscussion. Further experiments are needed to localize pre-cisely the regions of interactions and to determine the relativeaffinities of binding with the MetJ protein.Twenty base pairs after the stop codon of metC, there is a

region of dyad symmetry (Fig. 4). When transcribed, thisregion could form a stem and loop structure. However, it isnot followed by a stretch of thymines and thus does notcorrespond to the characteristic structure of a p-independenttermination signal (34).Comparison of f3-Cystathionase and Cystathionine -Syn-

thase. The complete amino acid sequences of the MetB- andMetC-encoded proteins have been compared and this re-vealed a significant homology. The alignment shown in Fig.

1000 1500 1900+

a:o 0 a-- c a:o:3 :3~ 008outF.~ LL LU ~ 3 LU LU () L8~~~~~~~~~a- ) Si 0c0 cS e ra>HSI I I ±41.o .$ olo , . J ao~om

- = - - _ ,..~~~ - - = _ ~ -

AStart codon

AEnd codon

FIG. 3. Restriction map and sequencing strategy for the metC gene. The 1.9-kb insert in plasmid pIP29 is shown with the restriction sitesselected for sequencing. The open and filled circles correspond to Sau3A and HincII sites, respectively. The arrows represent the direction ofsequencing by the Sanger method (20). The dashed lines indicate fragments sequenced according to the Maxam and Gilbert method (22). Thestart codon and end codon of the metC gene are shown. The boxes correspond to the pBR322 part of the pIP29 plasmid. The insert is limitedon the left (nucleotide 1) by the HindIII site of pBR322. On the right, the bacterial insert is not delimited by a restriction site, due to the artifactat the first step of the cloning (see text): the junction with pBR322 is at nucleotide 1666 of the Sutcliffe sequence (25).

Biochemistry: Belfaiza et al.

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870 Biochemistry: Belfaiza et al. Proc. Natl. Acad. Sci. USA 83 (1986)

AAGCTTTTGCTACCAAAATCAGCGGCGATATCGTTGGCCTGGTTTAAGACGCGCCTTCAGCCAGCAGTTGCTGCTCGCGCTTAAGGGACGCTTCTGATTCAAGACTCTACGCTCTTAC100

TGAAGAGATTGCCCAGGTGACTACGGAGGCCAAAATAAGCCCMATCATCACGCACTTAMCGACAATATCGGCGTGCTGATACATACCC'CAGACCGGAAGGTCCGTCTGCATTAAATTAT200

TACCCACTGTGTATCTCCAGGACGCAAGTCACAAATCTGCGCATAATATATCAAACGACGTCGAATTGATAGTCGTTCTCATTACTATTTGCATACTGCCGTACCTTTGCTTTCTT;300

TCCTTGCGTTTACGCAGTAAAAAGTCACCAGCACGCCATTTGCGAAAATTTTCTGCTTTATGCCAATTCTTCAGGATGCGCCCCGCGAATATTCATGCTAGTTTAGACATC CAGACGTA'T* * * 400

MletAlaAspLysLy sLeuAspThrGlnLeuVa lAsnAlaG lyArgSerLysLysTyrThrLeuG lyAlaValAsnSerVal I leGlnArgAlaSer SerLeuMAAAACAGGMTCCCGACATGCCGGACMAAAAGCTTGATACTCMACTGGTGMATGCAGGACGCAGCAAAAAMTACACTCTCGCCGCGGTAMATAGCCTGATTCAGCGCCCTTCTTCGCTG

500 . . . . . . . . . 600Va1PheAspSerVa lGluAlaLysLys~isAlaThrArgAsnArgAlaAsnGlyGluLeuPheTyrGlyArgArgGlyThrLeuThr~isPheSerLeuGlnGlnAlakletCysGluLeuGTCTTTGACAGTGTAGAAGCCAAAAAACACGCGACACGTMATCGCGCCMATGGAGAGTTGTTCTATGGACGGCGCGGMACGTTMACCCATTTCTCCTTACAACMAGCGATGKGTGAACTG

700G luG lyG lyAl~aG lyCysVal1LeuPheProCysG lyAlaAlaAlaValAlaAsnSer I leLeuAlaPhelIleG luG lnGlyAspHisVa lLeuMetThrAsnThrAlaTyrG luProSerGAAGGTGGCGCAGGCTGCGTGCTATTCCCTGCGGGGCGGCAGCGGTTGCTAATTCCATTCTTCCTTTTATCGAACAGGGCGATCATGTGTTGATGACCAACACCGCCTATGAACCGAGT

* * * * * * * 800.G1lnAspPheCysSerLy sIleLeuSerLysLeuG lyVal~hrThrSerTrpPheAspProLeuI leGly~laAspI leValLysHisLeuGlnProAsnThrLysI leVaIPbeLeuGluCAGGATTTCTGTAGCAAMATCCTCAGCAAACTGGGCGTMACGACATCATGGTTTGATCCGCTGATTGGTGCCGATATCGTTMAGCATCTGCAGCCAAACACTMAAATCGTGTTTCTGGAA

* * * * . 900 . *SerProG lySer I leThrMe tG luValHisAspValPro~laI leVa lAl'aAlaValArgSerVa lValProAspAla I lelI leMe t I1leAspAsnThrTrpAlaAlaG lyValLeuPheTCGCCAGGCTCCATCACCATGGAAGTCCACGACGTTCCGGCGATTGTTGCCGCCGTACGCAGTGTGGTGCCGGATGCCATCATTATGATCGACAACACCTGGGCAGCCGGTGTGCTGTTT

1000LysAlaLeuAspPheGlyI leAspVa lSerI leGlnAlaAlaThrLysTyrLeuValGly~isSerAspAlaMet I leGlyTbrAlaValCysAsnAla~rgCysTrpGiuGlnLeuAr;AAGGCGCTGGATTTTGGCATCGATGTTCTATTCAAGCCGCCACCAAATATCTGGTTGGGCATTCAGATGCGATGATTGGCACTGCCGTGTGCAATGCCCGTTGCTGGGAGCAGCTACGG

* 1100 * * ***... . 1200

llO0 . . . . . . . 130020G luAsnAlaTyrLeuMetGrlyGlnMetVallaA~espThrAlaTyr IleThrSerArgG lyLeuArghrLeuGplyVa lArgLeuArgG lnlispLis luSerSerLeAuLysValAlaGAAAATGCCTATCTGATGGGCCAGATGGTCGATGCCGATACCGCCTATATAACCAGCCGTGGCCTGCGCACATTAGGTGTGCGTTGCGTCAACATCATGAAAGCAGTCTGAAAGTGGCT

1300GluTrpLeuAlaGlu~i sProGlnValAlaArgValAsniisProAlaLeuProG lySerLysGl1y~isGluPheTrpLysArgAspPheThrC lySerSerG lyLeuPheSerPheVa 1GAATGGCTGGCACATCCGCAMGTTGCGCGAGTTAMCCACCCTGCTCTGCCTGGCAGTMAGGTCACGTTCTGGAMCGAGACTTTACAGGCAGCAGCGGGCTATTTTCCTTTGTG

1400LeuLysLysLysLeuAsnAsnG luGluLeuAlaAsnTyrLeuAspAsnPhe SerLeuPheSerMetAlaTyrSerTrpG lyGlyTyrGluSerLeu IleLeuAlaAsnG lnProG luHi sCTTAAGAAAAACTC;AATAATGAAGAGCTGGCGAACTATCTGGATAACTTCAGTTTATTCAGCATGGCCTACTCGTGGGGCGGGTATGAMTCGTTGATCCTGGCAAMTCAACCAGAACAT

1500I 1leAlaAla I 1eArgProGlnGlyGluI leAspPheSerGlyThrLeuI 1'eArgLeu~i sI leGlyLeuGluAspVa lAspAspLeuIl1eAlaAspLeuAspAlaG lyPheAlaArglIl1eATCGCCGCCATTCGCCCACAAGGCGAGATCGATTTTAGCGGCACCTTGATTCGCCTGCATATTGGTCTGGMAGATGTCGACGATCTGATTGCCGATCTGGACGCCGGTTTTGCGCGAATT

1600Val***GTATAACATTGCCACTTTTGGACATTTTGCAGACATTTTATTGTGAAAAGTCTTAAATTGTTGCGTCCGGGATCAAGGCGTCCCGGACGATTCAGGAGTACAATAGGCAGATAAAGGCT

1700 . . 1800TAACGCTGTTCCACAGGAMAGTCCATGGCTGTTATTCAAGATATCATCGCTGCGCTCTGGCMACACGACTTTGCCGCGC

FIG. 4. Sequence of the metC gene and its flanking regions. The deduced amino acid sequence of the MetC-encoded protein is indicatedabove the nucleotide sequence. The HindIII and EcoRI sites (see Discussion) are underlined and the putative ribosome binding site (26) isoverlined. The arrows indicate a region of dyad symmetry.

6 involves only 20 gaps for a total of 395 residues for the MetCprotein and 386 for the MetB protein; 126 amino acid residues(31%) are conserved between the two proteins. Furthermore,29 residues are accepted replacements (35), which gives a

total of 36% homology.From this comparison and the fact that this homology is

uniformly distributed throughout the two amino acid se-quences, it is most likely that the metB and metC genes haveevolved from a common ancestral gene. The original genemay have been duplicated with the copies ultimately residing

Table 2. Amino acid composition of ,3-cystathionase

Observed,*Amino acid mol per subunit

47

19

41

6

42

36

14

26

44

20

6

15

14

27

19

9

9

31

Predicted(from nucleotide

sequence)4318396

34301324401891812281969

29

-3A metc CGCGAATATTCATGCTAGTTTAGACATCCAGACGTATAAAAACAGGAATCCCG

* ** ** ** ****i** ****** **** * *-56metB AATCTATACGCAAAGAAGTTTAGATGTCCAGATGTATTGACGTCCATTAACAC

B metc

-50

ATATTCATGCTAGTTT A G A C A T C C 3A G A C G T A T AAAAACAGGAATCCCG

-119metB GGGATTTGCTCAATCT A T A C G C A A

A G A A G T T T

A GA T G T C C

A G A T G T A T -48

T G A C G T C C ATTAACACAATGTTTA-97

metF CGCCCTTCGGCTTTTC C T T C A T C T

T T A C A T C T

G G A C G T C T

A A A C G G A T -26

A G A T G T G C ACAACACAACATATAA

-67metA TTTTCTGGTTATCTTC A G C T A T C T

G G A T C T

A A A C G AT -4

A A G C G T A T GTAGTGAGGTAATCAG

R: A G AC G T C T

FIG. 5. Comparison of the 5' flanking regions of the met genes.(A) Comparison of the Met boxes of the metC and metB genes. Stars

between the nucleotide sequences indicate identical residues. Num-

bers indicate positions relative to the adenine of the respective start

codon taken as + 1. (B) Comparison of the 5' flanking regions of the

metC, -B, -F, -A genes. The sequences 5' to the structural metC,

metB, metF, and metA genes (this paper; refs. 11, 27, and 28) are

presented discontinuously and have been aligned in order to focus on

the presence of the underlying repetitive palindromic unit located

within the Met boxes. Bold characters indicate nucleotides matchingthe consensus sequence presented in line R. Numbers indicate

positions relative to the adenine of the respective start codon taken

as +1.

AlaArgAsxCysGlxGlyHisIleLeuLysMetPheProSerThrTrpTyrVal

*See ref. 17.

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Proc. Natl. Acad. Sci. USA 83 (1986) 871

C: HADKKLDTQLVNAGRSKKYTLGAVNSVIQRASSLVFDSV-EAKKHATRNRANGELFYGRR+* * * * * * * *+. * * + * * **

B MTRKQATIAVRSGLNDDEQYGCVVPPIHLSSTYNFTGFNEPRAHD----------YSRR

GTLTHFSLQQAMCELEGGAGCVLFPCGAAAVANSILAFIEQGDHVLMTNTAYEPSQDFCS* * +.* * ******* ** * *4+ *+. ** ++4 * *

GNPTRDVVQRALAELEGGAGAVLTNTGMSAIHLVTTVFLKPGDLLVAPHDCYGGSYRLFD

KILSKLGVTTSWF-DPLIGADIVKHLQPNTKIVFLESPGSITMEVHDVPAIVAAVRSVVP* * * * * + * *+.* +.*** * *+. * * *

S-LAKRGCYRVLFVDQGDEQALRAALAEKPKLVLVESPSNPLLRVVDIAKICHLAREVG-

DAIIMIDNTWAAGVLFKALDFGIDVSIQAATKYLVGHSDAMIGTAVCNARCWEQLRENAY*+. +*** + * * *+. + **** **** * * *+. + + * *

-AVSVVDNTFLSPALQNPLALGADLVLHSCTKYLNGHSDVVAG--VVIAKDPDVVTELAW

L---MGQMVDADTAYITSRGLRTLGVRLRQHHESSLKVAEWLAEHPQVARVNHPALPGSK* * *+. ****** * + * * * ++4 ** **

WANNIGVTGGAFDSYLLLRGLRTLVPRMELAQRNAQAIVKYLQTQPLVKKLYHPSLPENQ

GHEFWKRDFTGSSGLFSFVLKKKLNNEELANYLDNFSLFSMAYSWGGYESLILANQPEHI*** * * ** * * * ***+. * * ** ****

GHEIAARQQKGFGAMLSFELDGDEQT--LRRFLGGLSLFTLAESLGGVESLISHAATMTH

AAIRPQGEIDF--SGTLIRLHlGLEDVDDLIADLDAGFARIV* * * **+.*+ *+.** +.******+. **

AGMAPEARAAAGISETLLRISTGIEDGEDLIADLENGFRAANKG

FIG. 6. Comparison of the MetC- and MetB-encoded proteins.The whole amino acid sequences of the f3-cystathionase (C) andcystathionine y-synthase (B) are presented in the one-letter code andhave been aligned by introducing gaps (-) in order to maximizeidentities. Stars indicate identical residues and + signs indicateaccepted replacements (I-L-V, D-E, R-K, T-S).

at totally different sites on the E. coli chromosome. Subse-quently, mutations have occurred in the ancestral metB andmetC genes leading to specialization of the encoded proteins.Cystathionine y-synthase specifies a y replacement withO-succinylhomoserine and L-cysteine as substrates but isalso capable of /3 elimination with a very low efficiency (1).This "cystathionase" activity may represent a vestige of theancestral protein. According to a proposal put forward byHorowitz (36) 40 years ago, the biosynthetic pathways as weknow them today have been progressively built backwardsfrom the final metabolite of the pathway. In 1965, Horowitz(37) rejuvenated his theory based on gene duplication fol-lowed by mutation, mainly taking into account the hypothesisformulated by Lewis (38) ofgene duplication as an importantfactor of evolution. If the theory is correct, one expects thatthe products of the mutated copies may have retained amore-or-less pronounced homology with the product of theirancestral gene and between themselves. The results reportedhere may have some bearing on this theory. In addition, onemay wonder whether or not the common ancestor sequencecould extend to the regulatory region that is found highlyhomologous upstream from the metC and metB genes.

We thank James D. Ownby and Octavian Barza for critical readingof the manuscript and Philippe Marliere for helpful discussions. Weare grateful to M. Bruschi and M. Bonicel for the rapid and expertdetermination of the NH2-terminal sequence of the protein and toPatrick Trieu-Cuot and Gerlinde Lenzen for advice concerningnucleotide sequencing. We acknowledge Mathias Springer andRonald Greene for gifts of strains, Nicole Guiso for iodinatedmarkers, and Lucile Girardot for typing the manuscript. JamilaBelfaiza is the recipient of Moroccan and French fellowships. Thiswork was supported by grants ofthe Centre National de la RechercheScientifique (U.A. 1129), the Institut National de la Sante et de laRecherche Medicale (841006), and the Comite Consultatif des Ap-plications de la Recherche (Institut Pasteur, 7170).

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