Random mutagenesis of the zinc-binding motif of betaine-homocysteine methyltransferase reveals that Gly 214 is essential.
Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[ILV]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal.
Breksa
Andrew P
AP
Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801, USA.
Garrow
Timothy A
TA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D050940:Betaine-Homocysteine S-Methyltransferase; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D005998:Glycine / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D015032:Zinc / Q000378:metabolism
D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; D015032:Zinc; D005998:Glycine