Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

• DOI: 10.1128/jb.01094-07
• Publication Date: 2008-01-12T01:46:12Z
• Authors: Jonathan E. Ulmer; Yap Boum; Christopher D. Th...; Hannu Myllykallio; Carol Hopkins Sibley
• Keywords: Models, Molecular; Amino Acid Motifs; Molecular Sequence Data; 03 medical and health sciences; Bacterial Proteins; [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology; Escherichia coli; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology; Amino Acid Sequence; Amino Acids; [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology; 0303 health sciences; Molecular Structure; Sequence Homology, Amino Acid; Genetic Complementation Test; Mycobacterium tuberculosis; Thymidylate Synthase; Protein Structure, Tertiary; 3. Good health; [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology; Mutation; Flavin-Adenine Dinucleotide; Electrophoresis, Polyacrylamide Gel; Deoxyuracil Nucleotides; NADP; Thymidine

ABSTRACT

A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX 7-8 S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a Δ thyA strain of Escherichia coli . Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X 24 H69 X 25 R95HRX 7 S105 XRYX 90 R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.