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PubMed1998

Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.

Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.

Chromatography, Affinity↗
PubMed1996

The corrinoid-containing 23-kDa subunit MtrA of the energy-conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum. EPR spectroscopic evidence for a histidine residue as a cobalt ligand of the cobamide.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum is a membrane-associated enzyme complex that catalyzes an energy-conserving, sodium ion translocating step in methanogenesis from H2 and CO2. The complex is composed of eight different subunits, MtrA-H, one of which (MtrA) harbours a corrinoid as prosthetic group. In this study, we report the structural properties of MtrA1 [des-(214-239)-MtrA], which is a deletion mutant of MtrA that lacks the last 25 C-terminal hydrophobic amino acids rendering the membrane protein soluble: (a) mtrA1 was heterologously expressed in Escherichia coli. Overexpression yielded a cytoplasmic protein which was purified approximately tenfold to apparent homogeneity. The purified protein was devoid of its corrinoid prosthetic group and not correctly folded as was evident from its electrophoretic mobility in SDS/PAGE. (b) Unfolding of MtrA1 with guanidine/HCl and refolding in the presence of cobalamin resulted in the formation of the correctly folded MtrA1 holoprotein that contained tightly bound cob(II)-alamin; the rate of reconstitution was highest when the refolding proceeded in the presence of titanium(III) citrate, which suggested that cob(I)alamin is the corrinoid species that binds to the apoprotein. (c) EPR spectra of the cob(II)alamin-containing holoprotein differentially labelled with 14N (nuclear spin 1) and 15N (nuclear spin 1/2) revealed that the corrinoid is bound to MtrA1 in the base-off form and that the Co(II) of the prosthetic group is coordinated by a histidine residue of the apoprotein. The results are interpreted with respect to the mechanism of energy conservation by the MtrA-H complex.

Cloning, Molecular↗