In Methaizosarcina burkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT 1 and MT2. Transferase MTI is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R. K. (1996) ELK J. Riochem. 235, 653-6591, We report here on the corresponding analysis of MTl. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 pmol min-' mg-'. The enzyme catalyzed the methylation of its bound corrinoid in the cob(1)amide oxidation state by methanol. In addition to this autoinethylation, the purified enzyme was found to catalyze the methylation of free cob(1)alamin to methylcob(II1)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtuC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtuCB. The corrinoid-harbouring subunit MtaC exhibits 35 % sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

(Received 18 September 1Y9S) -EJB 95 151x14 ML.thLirio,rurr,inl1i brrrkpri is known to contain two tnethyltransferase isoenzymes, here designitted MtaA and MtbA, which catalyze thc formation of methyl-coen/,ynIe M from nicthy1cobal;itnin and coenzyme M. The ge,nes encoding the two soluble 34-kDa proteins havc bccn cloned and sequenced. m!uA and mtbA were found to be located in different parts of thc genome, each forming a rnonocystronic lranxcription unit. Northern blot analysis revealed that mtirA is preferentially trunscribed whcn M . harkeri is grown on methanol and the rnthA gene when the organism is grown on HJCO, or trimethylamir *; . Comparison of the deduced amino acid sequences rcvcaled the sequences of the lwo isocnzyincs to hc 37% identical. Both isoenzymes showed sequence similarity t o uropoiphyrinogcn I11 decarboxylase from E,s(h~richii~ coli. The mtuA gcnc wiis tagged with a sequcncc encoding six His placed six bp bcforc thc ~f u A start codon, and was functionally overexpressed in E. coli. 25%) of the E. r d i protein was found to be active tnethyltransferasc which could be, purified in two steps to apparenr hoinogciiity with a 70 % yield.Ke-vwords: methanogenic Archaea; Methariosarcirru hurkPri; melhyltransfclases ; corrinoids ; uroporphyrinogen 111.Fermentation of methanol to rnethanc and C 0 2 in Methanosurcinu hurkeri proceeds via methyl-coenzyme M as an intermediate. Methyl-coenzyme M formation from methanol and coenzyme M has been shown to be catulyzed by an enzyme system composcd of a heterodimcric protein (54 kDa + 29 kDa) containing a corrinoid as a prosthetic group, and a monomeric protein (34 kDa) without a cofactor. The heterodimeric protein, designated rrielhyltransferase 1 (MTI), catalyzes the methylation of its corrinoid prosthetic group with methanol, and the monomeric protein, designatcd MT2, catalyzes the transfer of the methyl group to coenzyme M. MT2 also mediates the formation of tnelhyl-coenzyme M from free methylcobalatnin and coenzyme M which can be exploited to assay its activity (van der Meijden et al., 19X3a,b,c, 1984a.b (Yeliseev et ul., 1993). Evidence was presented that one isoenLymc, hcre designated MtaA, is involved in methanol mctubolism and the olhcr isoenzyrne, here designated MtbA, is involved in iiicthylamine metabolism (Ycliseev et al., 1993;Burke and Krzycki, 1995).An cnzyme activity catalyzing methyl transfer from nicthylcobalamin to coenzyme M was first observed in Metl~Lanrnbacte-riirrir fhe~~1~11r11ltr)tr~)~~hicuni by Taylor and Wolfc (' I 974). The activity was later found to be associaled with a metnbraiie-bouii~l corrinnid-coniaii7ing multienzyme complcx (Mtr) catalyzing methyl transfer froin N-'-mcthyltetrahy~r~iiiietli~iiio~te~in to wenLyinc M (Giirtner et al., 1993). The energy-conserving coinplex, which is composed of eight different subunits (Harms ct d., 19?5), was shown to catalyze rhc incthylation of Gee cob(1)-nlamin with incthyltetrahyclroniethan~)ptel.it~ atid the dcniethylation of incthylcobalamin with coenzyme M (Giirtncr et al., 1994;...

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane-associated enzyme complex which catalyzes an energy-conserving, sodium-ion-translocating step in methanogenesis from H2 and CO2. We report here that the complex is composed of eight different subunits for which evidence was obtained at the protein, DNA and RNA levels: (a) SDS/PAGE of the purified complex revealed the presence of eight different polypeptides of apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB) and 12 kDa (MtrF). The N-terminal amino acid sequences of the 12-, 12.5- and 13-kDa polypeptides, which had previously not been accessible, were determined; (b) cloning and sequencing of the corresponding genes revealed the presence of the eight mtr genes organized in a 4.9-kbp gene cluster in the order mtrEDCBAFGH; (c) Northern-blot analysis revealed the presence of a 5-kbp transcript. DNA probes derived from the mtrE and mtrH genes hybridized to the transcript, indicating that the eight mtr genes are organized in a transcription unit. By primer extension, the 5' end of the mtrEDC-BAFGH mRNA was analyzed. The mtr operon was found to be located between the methyl-coenzyme M reductase I operon (mcr) and a downstream open reading frame predicted to encode a Na+/Ca2+, K+ exchanger.