Goodtzova K, Kanugula S, Edara S, Pegg A E
Department of Cellular and Molecular Physicology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Biochemistry. 1998 Sep 8;37(36):12489-95. doi: 10.1021/bi9811718.
Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.
酪氨酸 - 114是O6 - 烷基鸟嘌呤 - DNA烷基转移酶（AGT）所有已知序列中13个完全保守的氨基酸之一。通过将人AGT中的该氨基酸替换为苯丙氨酸（F）、丙氨酸（A）、苏氨酸（T）或谷氨酸（E），研究了该氨基酸在AGT修复烷基化DNA中的重要性。然后比较了突变蛋白与野生型蛋白在以下能力方面的差异：（a）保护大肠杆菌免受甲基化剂N - 甲基 - N'- 硝基 - N - 亚硝基胍（MNNG）的侵害；（b）体外修复甲基化DNA；（c）与含有O6 - 甲基鸟嘌呤的寡脱氧核苷酸结合；（d）与低分子量假底物O6 - 苄基鸟嘌呤反应。当在缺乏内源性AGT的大肠杆菌菌株GWR109中高水平表达时，野生型和Y114F突变体在减少MNNG引起的突变和细胞杀伤方面非常有效。Y114A突变体的保护作用要小得多，而Y114T和Y114E突变体则无活性。所有四种AGT突变体的纯化制剂与O6 - 苄基鸟嘌呤反应速率的降低程度大致相似（74 - 120倍）。相比之下，体外对甲基化DNA底物的活性降低程度因突变而异，较保守的Y114F仅导致30倍的降低，而Y114E最剧烈的变化则完全消除了活性。Y114A的变化导致1000倍的降低，而Y114T使活性降低了10000倍。所有突变都影响了AGT与含有O6 - 甲基鸟嘌呤的单链或双链寡脱氧核苷酸的结合。Kd增加的程度因氨基酸而异，分别增加了2 - 5倍（F）、7 - 11倍（A）、167 - 200倍（T）和600 - 1000倍（E）。这些结果与酪氨酸 - 114在AGT与其DNA底物的结合以及促进烷基转移中发挥作用一致。AGT可能与其他DNA修复蛋白类似，使目标碱基从DNA螺旋中“翻转出来”。因此，酪氨酸 - 114很可能在与翻转的O6 - 甲基鸟嘌呤的相互作用中起关键作用。结果还表明，当细胞中产生大量AGT时，AGT修复甲基化DNA效率的大幅降低并不妨碍其保护大肠杆菌免受有毒烷基化剂侵害的能力。活性降低了30倍的突变体Y114F在提供这种保护方面与野生型AGT相当。
