Functional analysis of the Mycobacterium tuberculosis FAD-dependent thymidylate synthase, ThyX, reveals new amino acid residues contributing to an extended ThyX motif.
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 delta 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)H69X(25)R95HRX(7)S105XRYX(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.
Ulmer
Jonathan E
JE
Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065, USA.
Boum
Yap
Y
Thouvenel
Christopher D
CD
Myllykallio
Hannu
H
Sibley
Carol Hopkins
CH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013936:Thymidine / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D003856:Deoxyuracil Nucleotides; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D013936:Thymidine