Characterization of Betaine:homocysteine methyltransferase, cystathionine beta-synthase and cystathionine [gamma]-lyase: proteins controlling homocysteine metabolism
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The objective of this dissertation was to produce three of the human homocysteine metabolizing enzymes, cystathionine â -synthase (CBS), cystathionine ã -lyase (CGL) and betaine:homocysteine methyltransferase (BHMT) for use in structural studies. A modified pET28b vector was designed to produce N-terminal poly-histidine tagged proteins with a simple construction scheme having broad applicability by the use of rare Sap I cloning sites. With CBS, the main focus was to address the aggregation problems of the full-length protein. After various futile attempts to solubilize CBS, denaturation and renaturation of the protein did yield soluble protein, but the protein was inactive. Finally, soluble, full-length and active CBS was obtained by the manipulation of factors such as pH, medium of growth and addition of detergent. Chromatography and SDS-PAGE analysis revealed the presence of an inseparable, proteolytically cleaved form of CBS along with the full-length protein, confirming the protein's strong tendency towards aggregation. The insolubility problems of CGL were tackled by the usage of the designed expression vector and modification of the induction-time. Preliminary X-ray studies of BHMT crystals revealed that BHMT is a homotetramer, which was confirmed by gel-filtration analysis. An earlier study of the activating effect of SAMe on rat BHMT was challenged when it was found that SAMe had no effect on the recombinant BHMT’s ability to methylate homocysteine, nor did the enzyme appear to bind SAMe when examined by microcalorimetry. Crystallization trials of both CGL and BHMT were marred by oxidation-sensitivity and reproducibility problems.