Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.
dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.
Barabás
Orsolya
O
Institute of Enzymology, Biological Research Center, Hungarian Academy of Science, Budapest, Karolina út 29-31, H-1113, Hungary.
Pongrácz
Veronika
V
Kovári
Júlia
J
Wilmanns
Matthias
M
Vértessy
Beáta G
BG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D004952:Esters / Q000737:chemistry*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000737:chemistry*; D011485:Protein Binding; D011755:Pyrophosphatases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014498:Uracil / Q000737:chemistry; D014867:Water / Q000737:chemistry
D004952:Esters; D008024:Ligands; D010710:Phosphates; D014867:Water; D014498:Uracil; D004247:DNA; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium