Year: 2010
Type: dissertation
Abstract: 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), from cob(II), to the active cob (I) form in MS.MSR and one of its FAD/NADPH binding domains were cloned and expressed as GST fusion proteins in Escherichia coli.Two expression plasmids for full-length MSR, which contain a N-terminal GST tag and a C-terminal (His) 6 tag, were constructed and validated by sequencing and restriction enzyme digestion and successfully expressed in E. coli and Pichia pastoris.Subsequently, crystals of the FAD/NADPH binding domain of MSR that diffracted to 1.9 Ă, both with and without NADP +-were produced and the crystal structures were solved by molecular replacement methods.In addition, the activation domain of human MS was expressed in E. coli and purified and the crystallization conditions were determined.Based on structural information, site-directed mutagenesis of the two positions Asp 652 and Trp 697 of MSR was performed.The variants D652A, D652R and D652N of the FAD/NADPH binding domain of MSR and the variants D652A, D652R, D652N, W696A and W697H of the full-length MSR were cloned and expressed in BL21 E. coli cells.These variants of MSR were purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography.The kinetic properties of these variants of MSR were investigated using steady-state kinetic studies, steady-state inhibition studies, stopped-flow pre steady-state kinetic and redox potential studies.Compared with the data for the wild type MSR, the turnover number of the variants all decreased, the catalytic ability became lower and the midpoint potential of the cofactor FAD shifted to a more positive potential.Both 2'5-ADP and NADP + were competitive inhibitors for variants of MSR.However, 2'5'-ADP was a relatively stronger inhibitor than NADP + .All the data on variants of MSR suggested the Asp 652 and Trp 697 residues are important structural and functional determinants in MSR, in particular related to coenzyme binding and hydride transfer.To examine their dynamic properties, EPR, ENDOR and ESEEM were 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 following the binding of 2', 5'-ADP, NADP and the activation domain of MS to further analyse conformational properties of MSR in solution.
Author: Lou Xiao
Language: English
Subfield: Nutrition and Dietetics
Field: Nursing
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W173307845