Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 Å resolution

Parthasarathy Sampathkumar, Stewart Turley, Jonathan E. Ulmer, Gun Rhie Ho, Carol Hopkins Sibley, Wim G J Hol

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1091-1104
Number of pages14
JournalJournal of Molecular Biology
Volume352
Issue number5
DOIs
StatePublished - Oct 7 2005
Externally publishedYes

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Thymidylate Synthase
Mycobacterium tuberculosis
Flavin-Adenine Dinucleotide
Bromodeoxyuridine
Escherichia coli
Essential Genes
Archaea
Enzymes
Eukaryota
Adenosine
Genes
Catalytic Domain
Carbon
Bacteria
Pharmaceutical Preparations
4,6-dinitro-o-cresol
2'-deoxyuridylic acid

Keywords

  • FDTS
  • M. tuberculosis
  • Thymidylate synthase
  • ThyX
  • TSCP

ASJC Scopus subject areas

  • Virology

Cite this

Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 Å resolution. / Sampathkumar, Parthasarathy; Turley, Stewart; Ulmer, Jonathan E.; Ho, Gun Rhie; Sibley, Carol Hopkins; Hol, Wim G J.

In: Journal of Molecular Biology, Vol. 352, No. 5, 07.10.2005, p. 1091-1104.

Research output: Contribution to journalArticle

Sampathkumar, Parthasarathy ; Turley, Stewart ; Ulmer, Jonathan E. ; Ho, Gun Rhie ; Sibley, Carol Hopkins ; Hol, Wim G J. / Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 Å resolution. In: Journal of Molecular Biology. 2005 ; Vol. 352, No. 5. pp. 1091-1104.
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