Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, produc ing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDC; from Escherichia coli strain B (1.60 Angstrom), its complex with uracil (1.50 Angstrom), and a second active-site complex with glycerol (1.43 Angstrom). These represent the first high-resolution struct ures of a prokaryotic UDG to be reported. The overall structure of the E, c oli enzyme is more similar to the human UDG than the herpes virus enzyme, S ignificant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and h uman enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring sma ller substrate-induced conformational changes to bring active-site groups i nto catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E, col i and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results. Pro teins 1999;35:13-24. (C) 1999 Wiley-Liss, Inc.