โ Scribed by H. J. Nanninga; H. Veldkamp
No coin nor oath required. For personal study only.
The methanol: coenzyme M (HS-CoM) methyltransferase system from Methanosarcina barkeri was resolved on a DE-52-cellulose column into two components, MT l and MT 2. MT 2 was purified further and catalysed the formation of CH3S-CoM from HS-CoM plus either methanol or CH3-B I 2-DMBI, a methyl-Bl2 compound containing dimethylbenzimidazole as the ct-ligand. The first activity depended on the presence of ATP plus MgCI 2, of H 2 and of crude extract or MT b whereas the latter activity was found to be independent of the addition of these components.
MT I is an oxygen-sensitive enzyme which is not yet purified further. Anaerobic preparations are brown but turn red under aerobic conditions. Spectral properties of MT I indicate the involvement of a cobalamin in its functioning.
Further evidence of a cobalamin involvement in the methyltransfer reactions was obtained by HPLC analysis of samples of MT t, incubated at 37C for 15 min with methanol, HS-CoM and ATP plus MgCI 2 in various combinations under H 2. After the incubation the samples were heated for 15 min at 80 C in the presence of 80% methanol and the supernatant obtained after centrifugation was analysed on a ktBondapak Cl8 column with a linear gradient of (~50% methanol in 25 mM acetate, pH 6.0. In the presence of either HS-CoM or HS-CoM plus methanol only an adduct between HS-CoM and BI2-HBI, a Bl2 compound containing 5-hydroxybenzimidazole as the ~-ligand, was formed. When also ATP was present CH3-B 12-HBI was formed.
When increasing amounts of MT 1 were incubated with [lgC]-methanol in the presence of ATP plus MgC12 under 50% H2/50 % N 2 for 5 rain at 37~ increasing amounts of label were found in the protein fraction, obtained by treatment with 80% cold methanol.
The methyltransfer from methanol to HS-CoM appears to be catalysed by two separate enzymes MT l and MT 2. MT 1 contains a cobalamin which is enzymatically methylated in the presence of ATP plus MgCI 2. MT 2 transfers the methyl moiety from the bound CH3-BI2-HBI to HS-CoM and as a result CH3S-CoM is formed.
Anaerobic treatment of wastewater of a potato-starch factory
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