Nucleic Acids Research, 2003, Vol. 31, No. 24 7216-7226
© 2003 Oxford University Press
Article |
Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition
Department of Microbiology and Cell Biology and 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560 012, India
*To whom correspondence should be addressed. Tel: +91 80 293 2686; Fax: +91 80 360 2697; Email: varshney{at}mcbl.iisc.ernet.in
Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (Vmax / Km) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were 
7- and 173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the –5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease.