Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.
dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, alpha,beta-imido-dUTP and Mg(2+) at 1.5A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K(d) for alpha,beta-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.
Varga
Balázs
B
Laboratory of Genome Metabolism and Repair, Institute of Enzymology, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary.
Barabás
Orsolya
O
Takács
Eniko
E
Nagy
Nikolett
N
Nagy
Péter
P
Vértessy
Beáta G
BG
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification; D005453:Fluorescence; D008274:Magnesium / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry / Q000235:genetics
C110398:2'-deoxyuridine 5'-imidotriphosphate; D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D014364:Tryptophan; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium