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

Abstract

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant 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 human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli 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.

Timing facilitated site transfer of an enzyme on DNA

“…(iii) Finally, the trap must not bind to DNA and interfere with the sliding pathway. In the case of hUNG, these trap criteria were met by the previously characterized active site inhibitor uracil (U) 26 , which has a K i = 0.3 mM 27 , solubility in water of at least 20 mM at 37 °C, and it does not interfere with binding of hUNG to nonspecific DNA (Supplementary Results, Supplementary Fig. 1).…”

Escherichia coli Uracil DNA Glycosylase: NMR Characterization of the Short Hydrogen Bond from His187 to Uracil O2

“…The fundamental observation from these studies, which was entirely unexpected from the crystal structures of UDG (19,20), is that the enzyme active site is constructed to significantly stabilize the uracil anion such that it exists as a stable species at neutral pH. This observation suggests several catalytic advantages.…”

Structural Characterization of a Mouse Ortholog of Human NEIL3 with a Marked Preference for Single-Stranded DNA

“…Single-stranded selective monofunctional uracil-DNA glycosylase (SMUG1), removes uracil and 5-hydroxymethyluracil (5-hmeU) more efficiently from duplex DNA than from single-stranded DNA (Haushalter et al, 1999; Kavli et al, 2002; Wibley et al, 2003). The uracil-DNA glycosylases (of the same UDG superfamily as SMUG1) exhibit robust activity on duplex DNA, and their activity on single-stranded DNA is slightly better than that on duplex DNA (Mol et al, 1995; Savva et al, 1995; Slupphaug et al, 1996; Xiao et al, 1999). Some Fpg/Nei glycosylases such as Escherichia coli Fpg (EcoFpg), can recognize 8-oxoG and AP sites in both single and double-stranded DNA.…”