TY - JOUR
T1 - Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 Å resolution
AU - Sampathkumar, Parthasarathy
AU - Turley, Stewart
AU - Ulmer, Jonathan E.
AU - Ho, Gun Rhie
AU - Sibley, Carol Hopkins
AU - Hol, Wim G.J.
PY - 2005/10/7
Y1 - 2005/10/7
N2 - A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M. tuberculosis ThyX (MtbThyX) enzyme complements the E. coli χ2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.
AB - A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M. tuberculosis ThyX (MtbThyX) enzyme complements the E. coli χ2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.
KW - FDTS
KW - M. tuberculosis
KW - TSCP
KW - ThyX
KW - Thymidylate synthase
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UR - http://www.scopus.com/inward/citedby.url?scp=24944591878&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2005.07.071
DO - 10.1016/j.jmb.2005.07.071
M3 - Article
C2 - 16139296
AN - SCOPUS:24944591878
SN - 0022-2836
VL - 352
SP - 1091
EP - 1104
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -