JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4637-4645 Vol. 173, No. 150021-9193/91/154637-09$02.00/0Copyright © 1991, American Society for Microbiology

Purification, Characterization, and Molecular Cloning of S-Adenosyl-L-Methionine:Uroporphyrinogen III Methyltransferase

from Methanobacterium ivanoviiF. BLANCHE,' C. ROBIN,2t M. COUDER,1 D. FAUCHER,3 L. CAUCHOIS,2t B. CAMERON,2 AND J. CROUZET2*

Unite de Biologie MoMeculaire, Institut des Biotechnologies,2 Departement de Chimie Analytique,l and Unite deBiochimie des Macromolecules, Institut des Biotechnologies,3 Rh6ne-Poulenc Rorer S.A., Centre deRecherche de Vitry-Alfortville, 13 Quai Jules Guesde B. P. 14, 94 403 Vitry sur Seine Cedex, France

Received 11 March 1991/Accepted 24 May 1991

An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified inMethanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had anapparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, asestimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III intoprecorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L.Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMTdid not show substrate inhibition at uroporphyrinogen Ill concentrations of up to 20 ,uM. Oligonucleotideprobes from limited peptide sequence information were used to clone the corresponding gene. The encodedpolypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMTstructural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.

Methanogenic bacteria synthesize particular types of cor-rinoids, mainly 5-hydroxybenzimidazolylcobamide (vitaminB12 factor III) (24, 33, 34, 42, 46) and Coa-t[-(7-adenyl)]co-bamide (pseudovitamin B12) (24, 43). Occurrence of cobina-mide (factor B) has also been reported in Methanosarcinabarkeri (29).Methanogenic bacteria are known to synthesize large

amounts of corrinoids. Values ranging from 0.1 to 1.4 ,uLmolg (dry weight)- 1 have been reported (25, 43). The latter valuecorresponds to a high level of corrinoid compared with thatin eubacteria such as Streptomyces, Bacillus, Rhizobium,Agrobacterium, or Pseudomonas spp., in which levels sig-nificantly lower (the higher values are 0.1 ,umol g [dryweight]-') have been observed (31). Only propionic acidbacteria have been shown to synthesize the same amount ofcorrinoids (31). Therefore, enzymes involved in corrinoidsynthesis in methanogenic bacteria might show improvedcatalytic properties compared with the ones from organismsproducing lower amount of corrinoids.We described the purification of Pseudomonas denitrifi-

cans S-adenosyl-L-methionine:uroporphyrinogen III methyl-transferase (SUMT) (6); the cloning of its structural gene,named cobA (8); and its nucleotide sequence (13). P. deni-trificans SUMT exhibits two properties which have beensuggested to play a regulatory role in cobalamin biosynthesis(6). The enzyme has a low catalytic efficiency (kcat = 38 h-1)and shows substrate inhibition at urogen III concentrationsabove 2 ,uM. In methanogenic bacteria, like Methanobacte-rium ivanovii, the regulatory features of the cobamide path-way are expected to be very different from those of P.denitrificans. The present study was undertaken to purify

* Corresponding author.t Present address: Institut de Recherche Jouveinal, Ddpartement

de Biotechnologies, 94265 Fresnes Cddex, France.t Present address: 5 bis, rue Pierre Curie, 10150 Pont-Sainte

Marie, France.

SUMT from a methanogenic bacterium, to study its basickinetic properties, and to clone its structural gene. M.ivanovii was chosen to carry this work out since it is easilycultivable (22) at a scale allowing enough cells to be obtainedfor the purification of an enzyme that should be present inlow amounts.

MATERIALS AND METHODS

General methods. Standard procedures for DNA isolation,manipulation, analysis, amplification, Southern blots, andcolony hybridization have been used (39). The procedures tomobilize plasmid DNA from Escherichia coli to P. denitrif-icans were already described (8). M. ivanovii genomic DNAwas purified as previously described (40). Nucleotide se-quence was performed with Sequenase version 2.0 (UnitedStates Biochemical Corporation) with ot-35S-dATP (Amer-sham) on plasmid DNA as previously described (10) or onsingle-strand DNA according to the manufacturer's specifi-cations and protocols.Media, bacteriological techniques, and chemicals. P. deni-

trificans and E. coli were grown in Luria broth (39) forroutine culturing. M9 medium (30) was supplemented whenrequired with 20 mg of L-cysteine liter-'. The growthtemperature was either 37°C for E. coli or 30°C for P.denitrificans. M. ivanovii was cultivated as previously re-ported (40) at 37°C in the complex medium described byWhitman et al. (45). E. coli strains were grown anaerobicallyin jars using the BBL GasPak anaerobic system (BectonDickinson and Co., Cockeysville, Md.).A 1-liter culture of E. coli B5548 with plasmid pXL1832 or

pUC13 was grown in LB medium supplemented with ampi-cillin up to an optical density of 1 (610 nm), and then the cellswere pelleted by centrifugation. Strain SC510 Rif' harboringplasmid pKT230 or pXL1841 was cultured with 30 ml of PS4medium in 250-ml Erlenmeyer flasks as already described(8).

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4638 BLANCHE ET AL.

TABLE 1. Bacterial strains and plasmids used

Strain, plasmid, or phage Relevant properties Reference or source

StrainsEscherichia coli

B5548 F- zlacUJ69 rpsL thi cysG44 relA From P. Cossart and B. Gicquel,obtained by P1 transduction of thecysG44 mutation from NF1400 intoMC4100 (11)

DH5a F- endAl hsdRJ7 supE44 thi-1 X- recAl gyrA96 Clontech Laboratory, Palo Alto, Calif.relAl, F80dlacZlAM15

MC1060 A(1acIOPZYA)X74 galU galK strA2 hsdR 9Pseudomonas denitrificansSC510 Rifr Rif' isolate (100 jig ml-') of SC510 5SBL25 Cobalamin-producing strain from which SC510 5

derives through mutagenesesMethanobacterium ivanovii DSM2611 22

Plasmids and phagespBSK+ Ampr ColEl, multicloning site Stratagene, La Jolla, Calif.pUC13 Ampr ColEl, multicloning site 48pKT230 Kmr RSF1010, carries the Mob locus of RSF1010 1pRK2013 Kmr ColE1, carries the tra genes of RK2 16pXL694 Ampr ColEl, Ptrp promoter vector followed by 15

cDNA human angiogeninpGlO M13 derived, part of M. ivanovii corA gene This studypXL1809 Ampr ColEl, corA under the control of Ptrp and This study

ribosome binding site clIpXL1841 Kmr RSF1010, corA under the control of Ptrp This studyM13mpl9 M13 derived, multicloning site 48

E. coli DH5(x cells were transformed when it was neces-sary to assay the lacZ ox-complementation to identify colo-nies harboring recombinant molecules; otherwise, MC1060was used.

Oligonucleotides primers and PCR. Oligonucleotides weresynthesized with a Biosearch 8600 automatic DNA synthe-sizer and purified by polyacrylamide gel electrophoresis(PAGE) under denaturing conditions as already described(15). Primers Al and A2 were derived from parts of theSUMT NH2-terminal sequence (VYLVG and PGDPE, re-spectively) on the coding strand (sense primers), as thefollowing degenerate 27-mer: 5' GCGCGAATTCGT(A,G,T)TA(T,C)(C,T)T(A,T)GT(A,T,G)GG(A,T)GC 3' and 5' CGCGGAATTCCC(A,T,C,G)GG(A,T,C,G)GA(T,C)CC(A,T,C,G)GA(A,G)(C,T)T 3', respectively. These two primers areflanked with an EcoRI restriction site (underlined). Primer B(antisense primer) was derived from the internal SUMTpeptide sequence (GTLEN) on the noncoding strand as thesubsequent degenerate 27-mer: 5' CGCGAAGCTT(G,A)TT(T,C)TC(T,A)A(G,A)(A,T,G)GT(A,T,C,G)CC 3', containingat its 5' end an Hindlll restriction site (underlined). Theseoligonucleotides were used in combination (primer Al or A2with primer B) to prime 30 cycles of enzymatic amplificationby the Taq DNA polymerase on 100 ng of total genomic DNAof M. ivanovii DSM2611 as previously described (38). Poly-merase chain reaction (PCR) was carried out on a Perkin-Elmer-Cetus DNA thermal cycler using the Taq DNA poly-merase from Cetus. On each cycle of amplification, thedenaturation, annealing, and polymerization steps were, re-spectively, carried out for 1 min at 95°C, 2 min at 38°C, and 3min at 72°C. PCR products obtained with primers A2 and Bwere then digested by EcoRI and HindlIl and separated byPAGE, and a 615-bp fragment was then purified by electroe-lution and cloned into the corresponding sites of M13mpl9.Several recombinant clones were sequenced.

Bacterial strains and plasmids and plasmid constructions.Bacterial strains and plasmids used in this study are de-scribed in Table 1.For pXL1809 construction, 40 jig of M. ivanovii genomic

DNA was digested with EcoRI and BglII; the fragmentswere then separated by agarose gel electrophoresis, andfragments in the range size of 3 to 3.5 kb were purified byelectroelution and cloned into EcoRI-BamHI-digestedpBSK+. To create pXL1832, a PCR of the pXL1809 se-quence with the primers 1277 (5' GGCCGAATTCATATGGTAGTTTATTTA 3') and 1278 (5' GGCCGAGCTCTATTACATAATT 3') was carried out in the same conditions as forthe enzymatic amplification of an internal fragment of M.ivanovii SUMT structural gene, except that only 20 cycleswere performed. The PCR products were digested by NdeIand SstI (primers 1277 and 1278 have on their 5' ends,respectively, NdeI and SstI restriction sites as underlined)and then ligated with the EcoRI-NdeI (120 bp)- and theEcoRI-SstI (3.1 kb)-purified fragments from pXL694 (15).The 1.5-kb EcoRI-BamHI-BamHI fragment from pXL1832,obtained by partial digestion with BamHI of EcoRI-linear-ized pXL1832, was then cloned into pKT230 digested withthe same restriction enzymes to give pXL1841.Enzyme assay and determination of SUMT properties.

SUMT activity was assayed in cell extracts as previouslydescribed (6), except that incubations were carried out at 100,uM S-adenosyl-L-methionine (SAM) and 1 jiM urogen II}.One unit of SUMT was defined as the amount of enzymenecessary to transfer 1 nmol of methyl group per h underthese conditions. Kinetic parameters (Km for urogen III andVmax values) were obtained from the primary plot of initialvelocity against urogen III concentration (from 0.05 to 2.0,uM) at 100 ,uM SAM. The curve was fitted to weightedexperimental data with Enzfitter, a nonlinear regression dataanalysis program (Elsevier Science Publishers BV, Amster-

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SUMT ACTIVITY IN M. IVANOVII 4639

dam, The Netherlands). Determinations of protein concen-trations, native molecular weight estimation, and sodiumdodecyl sulfate (SDS)-PAGE of purified SUMT were carriedout as previously described (14).

Reaction catalyzed by SUMT from M. ivanovii. PurifiedSUMT (41 ,ug; 63 U) was incubated anaerobically in the darkat 30°C for 4 h in 0.1 M Tris hydrochloride, pH 7.7,containing 5 mM dithiothreitol (DTT), 1 mM EDTA, 140 puM[methyl-3H]SAM (6.8 ,uCi p.mol-1), and 0.86 ,uM urogen IIIin a total volume of 350 ml. After incubation, tetrapyrrolicacids were trapped on a small DEAE-Sephadex A 25 column(5 ml of swollen gel) and were oxidatively esterified with 5%(vol/vol) sulfuric acid in methanol (18 h, 20°C). The methylester derivatives were extracted with dichloromethane, pu-

rified by thin-layer chromatography using standard methods(6), and identified as previously described (6).

Purification of SUMT from M. ivanovii. The pH of Trishydrochloride buffers was adjusted to 7.6 at 20°C. Allhigh-performance liquid chromatography separations were

performed at 20°C with a Gilson 305 gradient system (Gilson,Villiers LeBel, France).

(i) Preparation of a cell extract. Bacteria (12 g [wet weight]of cells of M. ivanovii) were resuspended in 80 ml of 0.1 MTris hydrochloride-5 mM DTT-1 mM EDTA (buffer A) andwere sonicated at 4°C with 30 20-s bursts from a Branson B30 ultrasonic disintegrator. Cell debris was separated bycentrifugation at 50,000 x g for 1 h, and the clear superna-

tant was passed through a column of DEAE-Sephadex A 25(1 ml of swollen gel) which had been equilibrated with bufferA.

(ii) Ammonium sulfate fractionation. The protein fractionprecipitating between 55 and 75% saturation with ammoniumsulfate was collected by centrifugation (10,000 x g for 30min) and resuspended in 6.0 ml of 0.1 M Tris hydrochloride-0.5 mM DTT-1.7 M ammonium sulfate (buffer B).

(iii) Hydrophobic interaction chromatography. The enzyme

solution was applied at a flow rate of 1.0 ml min-' onto a

Phenyl-Superose HR 10/10 column (Pharmacia) previouslyequilibrated with buffer B, and proteins were eluted at 2.0 mlmin-1 with a 70-ml linear decreasing gradient of 1.7 to 0 Mammonium sulfate in buffer B and immediately adjusted to25% (wt/vol) glycerol. Active fractions were pooled (SUMTwas eluted with approximately 0.6 M ammonium sulfate) andpassed through a column of Sephadex G-25 which had beenequilibrated with 0.1 M Tris hydrochloride-0.5 mM DTT-25% glycerol (buffer C).

(iv) First anion-exchange chromatography. The proteineluate was loaded on a Mono Q HR 5/5 column (Pharmacia)equilibrated with buffer C. Proteins were eluted at 1.0 mlmin-' with a 15-ml linear gradient of 0 to 0.4 M potassiumchloride in buffer C.

(v) Second anion-exchange chromatography. Fractions con-taining SUMT activity (eluted with 0.2 M potassium chlo-ride) were pooled and mixed with 1 volume of buffer C. Thesolution was applied to a Mono Q HR 5/5 column eluted at1.0 ml min-' with a 25-ml 0 to 0.3 M linear gradient ofpotassium chloride in buffer C. The fraction containingSUMT activity was concentrated to 200 ,u1 with a Centricon10 microconcentrator (Amicon).

(vi) Gel permeation chromatography. The protein solutionwas injected on a Bio-Sil SEC 250 column (Bio-Rad) elutedat 0.5 ml min-1 with 50 mM sodium sulfate-20 mM sodiumdihydrogen phosphate (pH 6.8)-0.5 mM DTT-25%- glycerol.SUMT was eluted as a symmetrical peak.

Protein sequencing. About 500 pmol of SUMT was sub-jected to microsequencing by using the sequential automated

TABLE 2. Purification of SUMT from M. ivanovii

VlAmt of Sp act Re- Purifi-Purification step Vol protein (U mg covery cation(M)(mg) of%)o-(fold)

Crude extact 92 731 0.337Ammonium sulfate 7.1 153 1.215 76 3.6

precipitatePhenyl-Superose eluatea 6.5 8.34 15.35 52 46First Mono Q 5/5 eluate 1.0 0.855 144 50 427Second Mono Q 5/5 1.6 0.252 422 43 1,252

eluateBio-Sil 250 eluate 1.0 0.061 1,537 38 4,561

' Assayed after Sephadex G-25 chromatography.

Edman degradation method with an Applied Biosystemsmodel 477 apparatus. The sequence NH2-VVYLVGAGPGDPELITLKAVNVLKXADVVL . . . was determined. In or-der to obtain an additional sequence, the same quantity ofprotein was submitted to complete trypsin digestion. Thefragments were then separated on a Brownlee RP C8 re-verse-phase high-performance liquid chromatography col-umn (2.1 by 10 mm; flow, 0.2 ml min-'), eluting with an80-min gradient of 0 to 50% (vol/vol) acetonitrile in 0.07%aqueous trifluoroacetic acid. A peptide eluted in a well-separated peak at 31% acetonitrile was sequenced, giving thesequence NH2-IITGTLENIAGK.

Nucleotide sequence accession number. The nucleotidesequence shown in Fig. 5 has been submitted to GenBankunder accession number M62874.

RESULTS

Characterization of SUMT from M. ivanovii. The purifica-tion of M. ivanovii SUMT is summarized in Table 2. Theenzyme was purified about 4,500-fold with a 38% yield.

(i) Identification of products of SUMT activity. Thesestudies were performed with a purified SUMT preparationwhich was homogeneous, as judged by SDS-PAGE, andshowed a single symmetrical peak when analyzed by gelfiltration high-performance liquid chromatography and oneclean sequence when subjected to NH2-terminal amino acidsequencing. The products of SUMT activity were identifiedas factor I octamethyl ester and sirohydrochlorin octamethylester after oxidative esterification, thus indicating thatSUMT from M. ivanovii catalyzes both methylations at C-2and C-7 of urogen III leading to precorrin-1 and precorrin-2(see structures in Fig. 1). No trimethylated pigment in adetectable quantity was obtained. The precorrin-1-to-pre-corrin-2 ratio of intermediates produced in incubation con-ditions described in Materials and Methods was approxi-mately 65:35, a value not very different from that (1:1)obtained in experiments with SUMT from P. denitrificans(6).

(ii) Mr determination. The enzyme, when subjected toSDS-PAGE, migrated as a single protein band with a molec-ular weight of 29,000 + 1,000 (Fig. 2). The Mr determinationof the native enzyme by gel filtration gave a value of 58,200+ 2,000, indicating, as for P. denitrificans SUMT, a ho-modimeric structure.

(iii) Kinetic parameters. The Km for urogen III was foundto be 52 + 8 nM, a value about 20-fold lower than thatobtained for the enzyme from P. denitrificans. The Vmaxvalue was 1,537 U mg-'. Unlike SUMT from P. denitrifi-

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4640 BLANCHE ET AL.

C02Hg r ~C02H

> ° CO2HH02C

H3C~ HN C2

002H NH HN

C02H CO2H

(2)

CO2CH3

_, ( CO2CH3H3CO2

H3C > N HN C2CH3

CO2CH3\ NH N A

C02CH3

CO2CH3 CO2CH3

(4)

CO2H

H3C, CO2H

H3C N HN `- CO2H

C02H NH HN

CO2HH H

C02H CO2H

(3)

CO2CH3

S H3CS CO2CH3

H3CO2C

H3C' N HN N- CO2CH3

NH N

C02CH3

C02CH3 CO2CH3

(5)FIG. 1. Structures. Compound 1, urogen III; compound 2, precorrin-1; compound 3, precorrin-2; compound 4, factor I octamethyl ester;

compound 5, sirohydrochlorin octamethyl ester.

cans, the enzyme from M. ivanovii did not show substrateinhibition at concentrations of up to 20 ,uM urogen III.

(iv) Absorption spectrum. In 0.1 M Tris hydrochloride-0.5mM DTT-25% glycerol, purified SUMT showed a typicalprotein spectrum with a single absorption maximum at 265nm, and no absorbance in the visible region. Assuming an Mrof 58,200, the molar extinction coefficient at 265 nm was 2.0x 105 M-1 cm-.Cloning of the M. ivanovii SUMT gene. From the NH2-

terminal peptide sequence (30 residues) and the sequence ofan internal fragment (12 residues) of the purified enzyme,three different oligonucleotides probes were designed. Prim-ers Al and A2 (sense primers) are, respectively, 256- and48-fold degenerate 27-mers derived from the N-terminalsequence; primer B3 (antisense primer) is a 96-fold degener-ate 25-mer derived from the sequence of the internal frag-ment. Primers A2 and B take into account the known codonusage in M. ivanovii (41). Primers Al or A2 could be usedwith primer B for a PCR with M. ivanovii genomic DNA forgenerating a probe specific for the SUMT structural gene as

already described for the cloning of a full-length porcineurate oxidase cDNA (26). Two PCRs with M. ivanoviigenomic DNA were performed with primers A2 and B(reaction 1) or primers Al and B (reaction 2). Those PCRproducts, when analyzed by PAGE, give fragments of 615and 240 bp for reaption 1 and 630 and 170 bp for reaction 2.The specific product of reaction 2 should have a size of 21 bp

A B

qnW -*--- 14.4

-o- 20.1

_o-mo -30

-{-- 43

FIG. 2. SDS-PAGE of purified SUMT. Lane A, pure SUMT (100ng); lane B, molecular weight markers (in thousands) (10 to 20 ng perband). Gels were stained with silver.

(1)

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SUMT ACTIVITY IN M. IVANOVII 4641

in excess of the one of reaction 1, since primers Al and A2correspond to N-terminal peptide sequences that are sevenresidues apart. The size difference is in the range of whatwas observed between the fragments of 615 and 630 bp. The615-bp fragment was cloned into the replicative form ofM13mpl9 phage. In the clones having the expected insert,the obtained sequence should be, just after the EcoRI site, asequence coding for the last 21 residues of the SUMT-determined NH2-terminal sequence, since in the senseprimer A2, the sequence corresponds to residues 9 to 14 ofthe sequence. For two of six clones, the forecasted sequencewas observed. This also allowed the identification of theundetermined residue corresponding to a lysine codon(AAA) in the DNA sequence. One replicative form was keptfor this study and named pG10.

Southern hybridization, using 32P-labelled pGlO as a probeagainst several digestions of M. ivanovii genomic DNA,revealed that a 3.2-kb EcoRI-BglII fragment hybridizes withthe probe (Fig. 3). It was also found that M. ivanoviiBglII-HindIII, BglII-KpnI, and BgllI-Pstl fragments of, re-spectively, 2.0, 5.8, and 1.9 kb also hybridized with theprobe (Fig. 3). The EcoRI-BglII-digested chromosomal frag-ments of 3.0 to 3.5 kb were purified by agarose gel electro-phoresis, ligated with EcoRI-BamHI-digested pBSK+, andintroduced into the E. coli strain DH5a. Among 800 recom-binant colonies, one showed a strong positive hybridizationsignal with pGlO insert as a probe. Restriction analysis of thecorresponding plasmid DNA (named pXL1809) indicatedthat a 3.2-kb fragment had been inserted (Fig. 4). SupercoilDNA sequencing with primer Al allowed reading of asequence that overlapped the one obtained from the se-quence of pGlO with the M13 -20 universal primer, indicat-ing that at least part of the SUMT structural gene wascloned.

Nucleotide sequence and sequence analysis of the geneencoding SUMT activity. The strategy for sequencing was toextend the sequence from the part encoding the NH2-terminal of the purified SUMT by supercoil DNA sequencingwith oligonucleotides specific for the sequence on bothstrands. The sequence of a 955-bp fragment, presented inFig. 5, was obtained. It contains an open reading frame(ORF) coding for a 231-amino-acid protein (Mr, 24,900),from base 34 (ATG) to 729 (TGA). The NH2-terminal se-quence of the encoded polypeptide is strictly identical to theamino acid sequences determined from the purified SUMT,except that the amino-terminal methionine has been re-moved. Removal of the amino-terminal methionine has beenproposed to occur when the penultimate amino acid is valine(4, 20). The SUMT internal amino acid sequence, obtainedafter trypsin digestion, corresponds in Fig. 5 to bases 634 to669; as expected, a basic amino acid (arginine) is foundbefore the trypsin cleaving site. No other ORF was found inthe sequence. The deduced polypeptide sequence was ana-lyzed with the Hopp and Woods program (21) and presentedthe characteristics of a soluble protein, as expected frombiochemical data.The sequence 5' GGTGA 3', complementary to the 3' end

of Methanobacterium 16S rRNA (2), is found 5 bp upstreamfrom the initiation codon. Only one gene from M. ivanoviihas been sequenced (41). This gene codes for a proteinhighly homologous to NifH (nitrogenase Fe protein) from thenitrogen-fixing archaebacteria. The A+T content of theORFni,fH is very close (63%) to that (62.8%) of the SUMTgene. The noncoding region has a higher A+T content(67.4%), and the average A+T content is 64%, which isconsistent with that (63%) of the M. ivanovii genome (3). The

1 2 3 4

58 -)

3.2 -

21.9

FIG. 3. Southern blots of restriction digests of M. ivanovii ge-nomic DNA after hybridization with 32P-radiolabelled pGlO plas-mid. Lanes: 1, HindIII-BglII; 2, KpnI-BglII; 3, EcoRI-BglII; 4,BglII-PstI. The sizes of the hybridizing fragments are indicated inkilobases.

codon usage of the sequenced gene is very similar to that ofORFnifH (41), with a preference for codons ending in A andU and little use for codons containing GC or CG (data notshown). This gene was named corA since it encodes anactivity that has been shown to be involved in corrinoidsynthesis in P. denitrificans (6).

Sequence homology with other proteins having SUMT ac-tivity. M. ivanovii CorA and P. denitrificans CobA proteinsequences were compared by using the Kanehisa program(23). A total of 40.4% strict identity between the two proteinswas found on most of their length (data not shown). CorAwas also shown to have more than 47% strict identity withBacillus megaterium SUMT (36) and 41.7% strict identitywith the carboxy-terminal domain of the E. coli CysGprotein (32), which has been shown to have SUMT activity(32, 44). Sequences homologies between these four proteinsare reported in Fig. 6. The recently determined sequence ofthe Salmonella typhimurium CysG protein (47) is not pre-sented in this figure, since it is very homologous to the E.coli enzyme (94% strict identity). Highly conserved domainsamong these SUMT proteins are observed; they are likely to

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4642 BLANCHE ET AL.

Avail

Aval

gT1T2T sIHilld

Separate digestions of pXL694 by EcoRI-Ndeland EcoRI -SstlPufication of the 120 bp EcoRI-Ndel and the3.1 kb EcoRI-Ssti fragments

ligation andtransformationInto MC 1060

Total digest by EcoRI and partial digestion byBamHIPurification of the 1.5 kb EcoRI-BamHI-BamHlfragment

Purification of the900 bp fragment

BamHI-EcoRl digestion

ligation andtransformation IntoMc 1060

Methanobacterium ivanovii corA gene

EtI Terminator region of the E. coli rrnB operon

EMM_ Human anglogenin cDNA

'Hhdiii

FIG. 4. Construction of plasmids pXL1809, pXL1832, anu pXL1841. See details in Materials and Methods.

EcoRI

Aval

(BgI I)

E¢oRI

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SUMT ACTIVITY IN M. IVANOVII 4643

ccataattettttataatttaaacggtgaacac ATG GTA GTT TAT TTA GTA GGT GCG GGT 60M V V Y L V G A G

CCA GGA GAT CCC GAA CTT ATC ACT CTC AAA GCT GTA AAC GTG TTA AAA AAA GCG 114P G D P E L I T L K A V N V L K K A

GAT GTT GTA CTG TAC GAC AAA CCT GCA AAT GAA GAA ATT TTA AAG TAT GCT GAA 168D V V L Y D K P A N E E I L K Y A E

GGT GCA AAA CTA ATA TAT GTC GGA AAA CAA GCA GGA CAT CAT TAC AAA TCT CAA 222G A K L I Y V G K Q A G H H Y K S Q

AAT GAA ATC AAT ACT CTT CTT GTT GAA GAA GCA AAA GAA AAT GAT TTA GTA GTA 276N E I N T L L V E E A K E N D L V V

CGC CTT AAA GGT GGA GAC CCC TmT GTA TmT GGA AGA GGA GGC GAG GAA ATT CTG 330R L K G G D P F V F G R G G E E I L

GCC CTT GTA GAA GAA GGA ATT GAT TTT GAG TTA GTT CCA GGG GTA ACT TCT GCA 384A L V E E G I D F E L V P G V T S A

ATT GGA GTT CCA ACA ACA ATT GGG CTT CCA GTr ACT CAT AGA GGT GTT GCA ACA 438I G V P T T I G L P V T H R G V A T

TCG TmT ACA GTT GTT ACA GGT CAT GAA GAC CCA ACA AAA TGC AAG AAA CAG GTA 492S F T V V T G H E D P T K C K K Q V

GGA TGG GAC TTT AAA GCA GAT ACT ATT GTA ATA CTT ATG GGT ATT GGA AAT TTA 546G W D F K A D T I V I L M G I G N L

GCT GAA AAT ACA GCA GAA ATT ATG AAA CAT AAA GAT CCT GAA ACT CCA GTT TGT 600A E N T A E I M K H K D P E T P V C

GTA ATT GAA AAT GGT ACG ATG GAA GGT CAA AGG ATA ATA ACG GGC ACA CTG GAA 654V I E N G T M E G Q R I I T G T L E

AAT ATA GCT GGA AAG GAT ATT AAA CCT CCT GCT TTA GTG GTA TTG GAA ATG TTG 708N I A G K D I K P P A L V V L E M L

I

4

* *** *

II1VLpw2 D ; PYIV3 LY K4 DII(G H

1 aK23 ;T

vPCVPG

* ***

IIIpGI AY

hIASGCSAYt

4 ILUkEtkPGIEG3STCA ATG TmT TTA AAG AAAS M F L K K

tgaatcaaatcagtggctgatctattaagaaggcaatatcatgaatg

gattagaaggtaaaaaaattgttataacaagacctgctgaaagggctaaagactcagttgaaatggtaaaat

773

845

FIG. 5. Nucleotide sequence of the 955-bp fragment. The posi-tion of the ORF along with the predicted amino acids sequence ofthe encoded polypeptide is indicated. Noncoding DNA is repre-

sented by lowercase letters. The putative ribosome-binding site isindicated by horizontal bars under the nucleotides.

reflect conserved regions necessary for SUMT activity andprobably contain amino acids of the active site. It is strikingthat most of the conserved regions [consensus sequences VXLVGAGPGDXXL(I/L)TL ADV,RLKGGDPF(V/I)FGRGGEE, and VPG(INV)T] are within the three re-gions of homologies among the known (CobA and CobI) andthe proposed (CobF, CobJ, CobL, and CobM) P. denitrifi-cans SAM methyltransferases of the cobalamin pathway(12), indicating that they must be of great relevance formethyltransferase activity (Fig. 6). However, no significanthomologies were found between SUMT consensus se-

quences (GXGPG and VXXXXXGDP) and the conservedsequences of 5-methylcytosine-forming methyltransferases(35), which are also SAM-dependent C-methyltransferases,suggesting that there is no conserved domain in the primarysequences of such proteins concerning either the active siteor a SAM-binding site.

In addition to the homologies between their primarysequences, SUMT from B. megaterium, M. ivanovii, and P.denitrificans have very similar molecular weights (25,800,24,900, and 29,200, respectively), which confirms that E. coliCysG protein (Mr, 49,928) carries another domain.

Expression of Methanobacterium SUMT activity in E. coliand P. denitificans. Plasmid pXL1809 was introduced intostrain B5548, an E. coli cysG mutant carrying the cysG44mutation (11) and was found not to complement this mutanton M9 medium without cysteine either aerobically or anaer-

obically. This result suggests that either M. ivanovii SUMTis not sufficiently expressed in E. coli or, as previouslyreported for the P. denitrificans CobA protein (13), thisenzyme cannot complement the cysG mutation since thisdefect might involve, in addition to a SUMT activity defi-

1 FL SSGLDIM IGAITANLIAG2LIIIKIT TL IEEH3 i4 F'7NPKSKHDAIK WSJ1IGPTYIXtAN LPYOQ2YH

1 GRSSPDEVAAGE?PA IV

2GMPP, SLIIIGR3 KDP IEIPALWMLS4 GYT IVDIVKEE)IEP1IV

FIG. 6. Alignments of amino acid sequences of P. denitrificansCobA (1), 17 to 244; E. coli CysG (2), 217 to 442; M. ivanovii CorA(3), 2 to 226; and B. megaterium CobA (4), 4 to 231. Similarities are

indicated as follows: =, same amino acid in all four sequences; -,

amino acids belonging to the same group (hydroxyl/small aliphatic,A, G, S, and T; acid and acid amide, N, D, E, and Q; basic, H, R,and K; aliphatic, M, I, L, and V; aromatic, F, Y, and W). Spacescorrespond to gaps in the alignments. Lines under I, II, and III referto regions of homology among P. denitrificans SAM methyltrans-ferases of the cobalamin pathway (12), named CobA, CobI, CobF,CobJ, CobL, and CobM. Stars under strictly conserved residuesindicate amino acids which are conserved in at least four of the fiveP. denitrificans methyltransferases.

ciency, a lack in precorrin-2 oxidation and iron chelation.The last two reactions might not be catalyzed by M. ivanoviiSUMT and are necessary for siroheme synthesis (27).A construction of pXL1832 was carried out, as shown in

Fig. 4, in which M. ivanovii SUMT structural gene was

linked to both the strong E. coli Ptrp promoter and theefficient ribosome binding site from the A clI gene (15),which was 9 bp upstream of the corA initiation codon.pXL1832-dependent SUMT expression was studied in strainB5548, which should allow the detection of a weak SUMTactivity in cell extracts. The strain harboring plasmidpXL1832 showed a 52-fold-higher SUMT activity than thecontrol strain (with plasmid pUC13) (310 versus 5.9 pmolh-1 mg of protein-', whereas SUMT activity of a cysG+ E.

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coli is between 60 and 160 pmol h-1 mg of protein-'),indicating that M. ivanovii SUMT is expressed from the E.coli transcription and translation signals. However, theexpression level was not sufficient to visualize the corre-sponding band when the total cellular proteins were ana-lyzed by SDS-PAGE (data not shown). Plasmid pXL1832,like pXL1809, was not able to complement the E. coli cysGmutant B5548, indicating that SUMT expression is notsufficient for complementation of the cysG mutation.The expression cassette was subcloned on a wide-host-

range incQ-derived vector (Fig. 4), giving plasmid pXL1841,which was introduced by conjugative transfer into P. deni-trificans SC510 Rif. SUMT activity was studied further inone transconjugant cultivated in PS4 medium, and a 17-foldincrease compared with that in the control strain SC510Rif'(pKT230) was found (1,700 versus 100 pmol h-1 mg ofprotein-1). This result established that plasmid pXL1841carries the expression signals allowing expression of the M.ivanovii SUMT structural gene in P. denitrificans. Whetheror not this expression is dependent upon E. coli Ptrp and theA cII ribosome binding site remains to be demonstrated;however, this result confirmed that the ORF found on thenucleotide sequence encodes a protein showing SUMTactivity.

DISCUSSION

We report in this study the purification of a SUMT activityfrom M. ivanovii. This enzyme catalyzes the same reactionsas the previously purified P. denitrificans SUMT enzymeinvolved in corrinoid synthesis (6). However, this newlydescribed enzyme did not show inhibition by its substrate,urogen III, a property which has been suggested to play aregulatory role in cobalamin synthesis in P. denitrificans. M.ivanovii SUMT had a kcatlKm of about 4.7 x 105 S-1,whereas the value of this parameter for the P. denitrificansenzyme was 1.1 x 104 s-1 (6). This 45-fold-higher value forthe specificity constant is mainly due to the very lowMichaelis constant of M. ivanovii SUMT for urogen III (52nM). It might reflect the adaptation of the enzyme to the lowintracellular level of urogen III in cultured cells (concentra-tions of <50 nM were currently measured) (17).The property of substrate inhibition reported for P. deni-

trificans SUMT was shown not to be restricted to the highlymutagenized cobalamin overproducing strain SC510. Puri-fied SUMT from SBL25, which derives from the wild-typeP. denitrificans (28) by only two mutageneses, was shown topresent a pattern of substrate inhibition quite similar to thatof SC510 Rif' (5). Moreover, purified SUMT from B. mega-terium, whose primary sequence has more than 40% strictidentity to both the M. ivanovii and the P. denitrificansSUMT, was also shown to be negatively regulated by urogenIII (36). This suggests that SUMT substrate inhibition is ageneral feature in aerobic eubacteria.The interesting property of the M. ivanovii enzyme, com-

pared with P. denitrificans SUMT, is the absence of sub-strate inhibition. The differences between the two enzymesis interpreted in view of the amount of corrinoids producedby these two organisms. The P. denitrificans strain, fromwhich the CobA protein has been purified, derives from astrain that has been isolated from poultry litter (28) andproduces 100 nmol of cobalamin liter-' per g of dried cell.P. denitrificans is a gram-negative aerobic rod that shouldrequire cobalamin at very low level, as does E. coli, forwhich it is admitted that 25 molecules of cobalamin per cellare sufficient to support growth, in a metE background (7).

One way to limit cobalamin synthesis would be to negativelyregulate the enzyme activity in the first step by urogen III,when this substrate accumulates above a certain concentra-tion. On the contrary, methanogenic bacteria synthesizemore corrmnoids than wild-type P. denitrificans and othereubacteria (see Introduction) for unknown reasons. There-fore, it is consistent that the activity of the key enzyme fortheir corrmnoids pathways does not exhibit a regulation.

Furthermore, F430 must be synthesized in high amounts,in methanogenic bacteria, since it is the prosthetic group ofa protein constituting about 10% of the cell protein content(37). F430, the nickel-containing uroporphinoid prostheticgroup of methyl-CoM methylreductase component C (19),combines structural elements of both the comns and theporphyrins and contains two methyl groups at positions C-2and C-7 (18); it has been proposed to be synthesized fromprecorrin-2 (18). In this instance, corrmnoids and F430 wouldshare the same biosynthetic pathway up to precorrin-2, andSUMT would operate on both pathways. The properties ofM. ivanovii SUMT reflect this requirement for high levels ofcorrinoid and F430 synthesis.

ACKNOWLEDGMENTS

We express our gratitude to J. Lunel and J.-F. Mayaux for theirsupport during this work. We thank the CITI2 (Centre de TraitementInteruniversitaire d'Informatique a Orientation Biomedicale, Paris,France) for nucleic and protein sequences analysis programs. Wethank F. Cuine for the NH2-terminal sequencing of M. ivanoviiSUMT and T. Ciora for oligonucleotide synthesis. We acknowledgeL. Sibold, J.-P. Aubert, and M. Henriquet from the Pasteur Insti-tute, without whom this work would not have been possible, and weare particularly grateful to L. Sibold, who has since died. We thankR. Longin and his team for the cultivation of M. ivanovii.

This work was supported by both Rh6ne-Poulenc Rorer S.A. andRh6ne-Poulenc S.A.

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