Oligomerization is required for betaine-homocysteine S-methyltransferase function.
Betaine-homocysteine methyltransferase (BHMT) is a member of a family (Pfam 02574) of zinc- and thiol/selenol-dependent methyltransferases. All family members purified to date are monomers, except BHMT, which is an oligomer. We have studied how C-terminal truncation or mutagenic replacement of residues within or associated with the unique dimerization arm of this enzyme affects oligomerization and function. Two C-terminal truncation mutants, S325 and D371, do not express well in Escherichia coli and are inactive. Residues within the dimerization arm (H338, R346, W352, R361, P362, Y363, N364, and P365) and one that forms a hydrogen bond to the arm (E266) were changed to alanine. All mutants maintained a normal or near-normal ability to bind zinc. E266A, R361A, P362A, Y363A, N364A, and P365A displayed near-normal catalytic activity, but H338A had only 10% of the wild-type enzyme activity. Like the wild-type enzyme, most mutants eluted as tetramers from gel filtration columns and formed discrete bands on SDS-PAGE gels following glutaraldehyde crosslinking. Mutants R346A and W352A had negligible activity, eluted as dimers, and displayed aberrant crosslinking properties. These data indicate that unlike other Pfam 02574 members, oligomerization of BHMT is required for function.
Szegedi
Sandra S
SS
Division of Nutritional Sciences, Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801, USA.
Garrow
Timothy A
TA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001622:Betaine / Q000378:metabolism; D050940:Betaine-Homocysteine S-Methyltransferase; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D001622:Betaine; D008780:Methyltransferases; D050940:Betaine-Homocysteine S-Methyltransferase; D015032:Zinc; D005976:Glutaral