Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution.
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 chi2913 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.
Sampathkumar
Parthasarathy
P
Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
Turley
Stewart
S
Ulmer
Jonathan E
JE
Rhie
Ho Gun
HG
Sibley
Carol Hopkins
CH
Hol
Wim G J
WG
D000595:Amino Acid Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
D003067:Coenzymes; D003856:Deoxyuracil Nucleotides; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase