Human methionine synthase reductase (MSR) is a 78-kDa diflavin enzyme involved in folate and methionine metabolism. It regenerates the cofactor of methionine synthase (MS), cob(II), to reduce inactive MS. MSR and one of its the FAD/NADPH binding domain were cloned as GST-tagged fusion proteins for expression and purification in Escherichia coli. And a 1.9 Å Crystals of the FAD/NADPH binding domain of MSR with and without NADP+ were produced and carried out X-ray diffraction experiment and the structure of the crystal was solved by molecule replacement method. The activation domain of human MS was also expressed and purified in Escherichia coli and crystallization conditions determined. A new expression vector for full-length MSR, which contains a N-terminal GST tag, and C-terminal 6× His tag, was constructed and validated by sequencing, restriction enzymes digestion and successfully expressed in E. coli and Yeast Pichia pastoris. Based on the structure information, site-directed mutagenesis on the two positions Asp652 and trpytophan697 of MSR were designed and completed. The variants D652A, D652R, D652N of the FAD/NADPH binding domain of MSR and the variants D652A,D652R,D652N, W696A,W697H of the full-length MSR were cloned and expressed in BL21 (E. coli). The proteins of these mutants were purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography. And the kinetic studies on these variants of MSR were investigated in steady state kinetic study, steady state inhibition studies, stopped-flow pre steady-state kinetic and redox potential studies. Compared with the data of the wild type MSR, the turnover number of variants all decreased, the catalytic ability become lower and the midpoint potential of cofactor FAD occurred positive shift. Both 2'5-ADP and NADP+ were competitive inhibitors for variants of MSR. However, 2'5'-ADP was relative strong inhibitor than NADP+. All the data on variants of MSR suggested the Asp652 and tryptophan697 were two important structural and function determinant of MSR. To investigate the dynamic properties of EPR, ENDOR and ESMME are used to investigate the existence of the semiquinone flavin cofactors, FAD and FMN, and the hyperfine coupling arising from the interaction of some nuclei with the unpaired electron spin. ELDOR spectroscopy was applied to measure the distance between the FAD and FMN in MSR under the binding of 2', 5'-ADP, NADP and the activation domain of MS to further check the conformational change of MSR.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:529220 |
| Date | January 2010 |
| Creators | Lou, Xiao |
| Contributors | Scrutton, Nigel |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/biochemical-and-structural-studies-of-human-methionine-synthase-reductase(822952fc-8bef-4a30-9221-7fb154638193).html |
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