Graduate School of Science and Engnieering, Iryo Sosei University, Iwaki, Fukushima, Japan.
Graduate School of Life Science and Technology, Iryo Sosei University, Iwaki, Fukushima, Japan.
Acta Crystallogr F Struct Biol Commun. 2021 Dec 1;77(Pt 12):444-451. doi: 10.1107/S2053230X21011055. Epub 2021 Nov 11.
O-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13-2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O-methyl-2'-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix-turn-helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.
O-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）可去除鸟嘌呤碱基上的细胞毒性 O-烷基加合物，并防止细胞受到烷化剂诱导的基因组损伤。尽管有报道称对酪氨酸残基发生突变的酶的活性进行了计算研究，但尚未发现有关其突变体晶体结构的研究。在这项研究中，通过比较突变体及其与底物类似物的复合物的晶体结构，详细研究了 Tyr91 的功能。在这项研究中，将保守的氨基酸酪氨酸突变为苯丙氨酸，产生 Y91F 突变体，将负责接收活性位点中甲基的半胱氨酸突变为丝氨酸，产生 C120S 突变体，从而产生突变体。还创建了 Y91F/C120S 双突变体 StoMGMT。基于 Y91F 突变体 StoMGMT 的晶体结构讨论了酪氨酸的功能。StoMGMT 的晶体结构在 1.13-2.60Å 的分辨率下确定。除了突变部分之外，它们没有结构变化。在浸入 O-甲基-2'-脱氧鸟苷的 Y91F 突变体晶体的结构中，没有观察到脱氧鸟苷或甲基的电子密度，野生型 StoMGMT 中的基团也没有被氧化。因此，Tyr91 的羟基可能会阻止氧化剂进入活性部位。这表明，在物种之间的螺旋-转角-螺旋模体的 N 末端高度保守的酪氨酸保护了 MGMT 的活性部位，这些活性部位在仅修复一个烷基加合物后就会失活。总的来说，这些结果可能为理解高度保守氨基酸在确保自杀酶完整性以及促进其活性方面发挥作用的分子机制提供依据。
