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Abstract:
Methanobacterium thermoautotrophicum is a methane-forming archaeon that
grows on H-2 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 approximate to 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 K-m 0.1 mM). The enzyme was
specific for L-homocysteine as methyl acceptor. Methylcobalamin could be
substituted with methylcobinamide as methyl donor.
