Xiao G, Tordova M, Jagadeesh J, Drohat A C, Stivers J T, Gilliland G L
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute for Standards and Technology, Rockville, 20850, USA.
Proteins. 1999 Apr 1;35(1):13-24.
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.
DNA修复酶尿嘧啶DNA糖基化酶（UDG）催化从单链或双链DNA中水解诱变前的尿嘧啶残基，产生游离尿嘧啶和无碱基DNA。在此，我们报道了来自大肠杆菌B菌株的游离UDG（分辨率为1.60 Å）、其与尿嘧啶的复合物（分辨率为1.50 Å）以及与甘油的第二个活性位点复合物（分辨率为1.43 Å）的高分辨率晶体结构。这些是首次报道的原核UDG的高分辨率结构。大肠杆菌酶的整体结构与人类UDG比与疱疹病毒酶更为相似。在假定的广义酸（His187）和碱基（Asp64）的侧链位置上，细菌和病毒结构之间存在显著差异，这与之前在病毒和人类酶之间观察到的差异类似。总体而言，与病毒和人类酶相比，包含His187的活性位点环似乎预先形成了结构，需要较小的底物诱导构象变化就能将活性位点基团带入催化位置。这些结构差异可能与大肠杆菌和病毒酶在尿嘧啶识别机制上的巨大差异有关。野生型UDG以及D64N和H187Q突变酶的k(cat)对pH的依赖性与Asp64的广义碱催化一致，但没有提供存在广义酸催化剂的证据。针对这些结果对UDG的催化机制进行了批判性讨论。
