Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/60334
標題: Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078
作者: Kan, S.C.
許文輝
Liu, J.S.
Hu, H.Y.
Chang, C.M.
Lin, W.D.
Wang, W.C.
Hsu, W.H.
關鍵字: Corynebacterium glutamicum
Thymidylate synthase A
Thymidylate synthase
X
FAD binding
Site-based mutation
catalytic mechanism
functional-analysis
crystal-structure
complementing protein
active-site
drug design
thyx
refinement
resolution
substrate
期刊/報告no:: Biochimica Et Biophysica Acta-Proteins and Proteomics, Volume 1804, Issue 9, Page(s) 1751-1759.
摘要: The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His(6)-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414 mU mg(-1) protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3 angstrom resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70-73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX. (C) 2010 Elsevier B.V. All rights reserved.
URI: http://hdl.handle.net/11455/60334
ISSN: 1570-9639
文章連結: http://dx.doi.org/10.1016/j.bbapap.2010.05.006
Appears in Collections:分子生物學研究所

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