Expression of recombinant human betaine: homocysteine S-methyltransferase for x-ray crystallographic studies and further characterization of interaction with S-adenosylmethionine.
Elevated homocysteine as a result of dysfunctional metabolic enzymes is an independent risk factor for arteriosclerosis. Betaine:homocysteine S-methyltransferase (BHMT) (EC 2.1.1.5) is an important enzyme in the pathway of homocysteine metabolism in that it recycles methionine from homocysteine and nonfolate methyl donors. To initiate X-ray crystallographic structural studies, we created a BHMT expression construct for use in Escherichia coli that has a polyhistidine purification tag with no extraneous protein, usually found in commercial vectors, between the tag and protein sequence. The extra amino acids can hinder the crystallization process. A modified pET28b vector was designed to produce N-terminal polyhistidine-tagged proteins with a simple construction scheme having broad applicability because of the use of rare SapI cloning sites. BHMT expressed using this vector could be rapidly purified using metal chelate chromatography. Gel exclusion chromatography analysis showed that recombinant polyhistidine-tagged human BHMT is a tetramer. S-Adenosylmethionine (SAMe) has no effect on the recombinant BHMT's ability to methylate homocysteine nor does the enzyme appear to bind SAMe when examined by microcalorimetry.
Bose
Nandita
N
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, Athens, Georgia 30605, USA.
Greenspan
Phillip
P
Momany
Cory
C
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001622:Betaine / Q000737:chemistry* / Q000378:metabolism*; D050940:Betaine-Homocysteine S-Methyltransferase; D002151:Calorimetry; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D050904:Homocysteine S-Methyltransferase; D006801:Humans; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000737:chemistry*
D000596:Amino Acids; D018076:DNA, Complementary; D011994:Recombinant Proteins; D001622:Betaine; D006639:Histidine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D050904:Homocysteine S-Methyltransferase; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase