Methylcobalamin: coenzyme M methyltransferase isoenzymes MtaA and MtbA from Methanosarcina barkeri. Cloning, sequencing and differential transcription of the encoding genes, and functional overexpression of the mtaA gene in Escherichia coli.
Methanosarcina barkeri is known to contain two methyltransferase isoenzymes, here designated MtaA and MtbA, which catalyze the formation of methyl-coenzyme M from methylcobalamin and coenzyme M. The genes encoding the two soluble 34-kDa proteins have been cloned and sequenced. mtaA and mtbA wee found to be located in different parts of the genome, each forming a monocystronic transcription unit. Northern blot analysis revealed that mtaA is preferentially transcribed when M. barkeri is grown on methanol and the mtbA gene when the organism is grown on H2/CO2 or trimethylamine. Comparison of the deduced amino acid sequences revealed the sequences of the two isoenzymes to be 37% identical. Both isoenzymes showed sequence similarity to uroporphyrinogen III decarboxylase from Escherichia coli. The mtaA gene was tagged with a sequence encoding six His placed bp before the mtaA start codon, and was functionally overexpressed in E. coli. 25% of the E. coli protein was found to be active methyltransferase which could be purified in two steps to apparent homogeneity with a 70% yield.
Harms
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U
Max-Planck-Institut für terrestrische Mikrobiologie und Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany.
Thauer
R K
RK
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017021:Methanosarcina barkeri / Q000201:enzymology / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D004269:DNA, Bacterial; D007527:Isoenzymes; D008780:Methyltransferases; C023894:methylcobalamin-coenzyme M methyltransferase; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases