Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.
Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.
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Max-Planck-Institut für terrestrische Mikrobiologie, Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany.
Thauer
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D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019766:Genes, Archaeal; D007700:Kinetics; D046911:Macromolecular Substances; D017014:Methanobacterium / Q000201:enzymology* / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008780:Methyltransferases; C121199:methylcobalamin - homocysteine methyltransferase