Tchou J, Bodepudi V, Shibutani S, Antoshechkin I, Miller J, Grollman A P, Johnson F
Department of Pharmacological Sciences, State University of New York at Stony Brook, New York 11794-8651.
J Biol Chem. 1994 May 27;269(21):15318-24.
The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities. This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis. Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine. The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM). Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM). Km and Vmax were determined for the cleavage reaction. Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively. 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3). Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases. An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions. A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme.
大肠杆菌的8-氧代鸟嘌呤-DNA糖基化酶，也被称为甲酰胺基嘧啶-DNA糖基化酶（Fpg蛋白），具有N-糖基化酶和AP裂解酶活性。该酶通过有效地从DNA中去除甲酰胺基嘧啶损伤和8-氧代鸟嘌呤残基来修复氧化性DNA损伤。使用含有各种8-氧代嘌呤的特定寡脱氧核苷酸来检测Fpg蛋白的底物特异性，并确定DNA中官能团在损伤识别和催化中的作用。确定了Fpg蛋白对含有8-氧代-2'-脱氧鸟嘌呤、8-氧代-2'-脱氧腺嘌呤、8-氧代-2'-脱氧新蝶呤、8-氧代-2'-脱氧肌苷、无碱基位点以及C8-氨基芴鸟嘌呤的开环加合物的双链寡脱氧核苷酸的结合亲和力。8-氧代-dG:dC的C8酮基位于大沟中，与紧密结合相关（Kd = 8.9 nM）。当C8酮基位于小沟中时，如8-氧代-dG:dA（Kd = 340 nM），结合则弱得多。测定了切割反应的Km和Vmax。对于含有C6和C8酮基的8-氧代嘌呤的寡脱氧核苷酸双链体，特异性常数（Kcat/Km）始终较高，如8-氧代-dG:dC和8-氧代-dI:dC，其Kcat/Km分别为9.3和18 min-1 nM x 10(-3)。8-氧代-dN:dC缺乏C6酮基；特异性常数为0.024 min-1 nM x 10(-3)。综上所述，我们的数据表明8-氧代脱氧鸟嘌呤的C8酮基和甲酰胺基嘧啶的羰基使Fpg蛋白能够识别并结合含有这些修饰碱基的双链DNA。涉及底物C6酮基的酶催化反应导致这些损伤的去除。提出了一种涉及8-氧代鸟嘌呤O-6质子化的机制来解释该酶的N-糖基化酶活性。
