Document Type
Poster
Publication Date
8-1-2008
Abstract
Betaine homocysteine methyltransferase-2 (BHMT-2) is a zinc metalloenzyme that plays an important role in the regulation of the homocysteine metabolism. It uses S-methylmethionine (SMM) as a methyl donor for the methylation of homocysteine. BHMT-2 is a 73% homologous to the protein BHMT, which catalyzes the remethylation of betaine and homocysteine to form dimethylglycine and methionine. BHMT is abundant in the human liver, kidney, and lens of the eye. BHMT (and likely BHMT-2) are involved in regulating homocysteine and S-adenosyl-L-methionine (SAM, AdoMet) levels. The goal of this study was to generate recombinant BHMT-2 for structural and functional studies. Escherichia coli BL21 (DE3), were transformed with the expression construct coded for BHMT2, fused with a N- terminal His-tag, and then were grown. Recombinant BHTM-2 protein was purified using 6xHis-tag affinity chromatography. Also, size exclusion chromatography was used to purify His-BHMT-2. The dimer form of His-BHMT-2 protein was obtained as a result in the study. By the analyzed results of this study, the protein BHMT-2 can be purified by different methods of chromatography. BHMT-2 polymorphisms may contribute to BHMT-2 enzyme variants with different levels of activity, thus leading to different levels of SAM (aka AdoMet), which influences acetaminophen sensitivity/response and thus liver toxicity. However, further experiments are needed to make structural studies (crystallography) and functional (enzyme assays) to compare the WT and variant enzymes (corresponding to genetic polymorphisms in the gene). It is very important to make further studies in way to determine the mechanism and function of this protein in humans.
Keywords
crystallography, BASE protein--human, BHMT protein--human, BHMT2 protein--human, chromatography
Publication Title
Intersections Source Symposium 2008
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License
Department/Center
Biochemistry
Recommended Citation
Cruz, O. H., Wilk, D., Weinshilboum, R.M., & Yee, V. (2008). Expression and Purification of Human Betaine-Homocysteine Methyltransferase 2 (BHMT-2). Intersections Source Symposium 2008.