Department of Biotechnology, National Institute of Technology, Warangal-506004, India.
Department of Computer Science and Engineering, Alagappa University, Karaikudi-630003, India.
Curr Pharm Biotechnol. 2021;22(15):2071-2084. doi: 10.2174/1389201022666210218194653.
Human glutathione S-transferases (hGSTs) are phase-II detoxification enzymes that catalyze the conjugation of electrophilic compounds and glutathione. Anomalous excess production of NO in the cellular environment under diseased or stressed condition results in lethal effects to the cell. Studies have reported that the evolution of tyrosine-based GSTs as a defense mechanism by the cell to mitigate Nitric Oxide (NO) toxicity. The dual role of hGSTP1 as NO carrier and scavenger is a prelude for the research forthwith.
A plausible role of hGSTM1 as NO carrier is considered. Being a prominent cellular messenger and secondary metabolite, excess production of NO is lethal to the cell. Moreover, hGSTM1 polymorphisms lead to diminished catalytic activity that promotes a diseased state. Hence, it is compelling to generate hGSTM1 mutants that have more catalytic efficacy compared to Wild Type (WT).
hGSTM1 mutants with enhanced efficiency were generated using in silico and in vitro Site-Directed Mutagenesis (SDM). WT and mutant proteins were overexpressed and purified using affinity chromatography. The catalytic activity and binding efficiency of WT and mutant proteins towards CDNB (1-chloro-2, 4-dinitrobenzene) & NO were determined.
NO assay reveals the probable interaction of WT hGSTM1 with NO. In silico, SDM studies provided E129K and Q109K mutants with superior NO binding efficiency as compared to WT. The catalytic activity (GST and NO assays) of the mutants corroborate the in silico results.
WT hGSTM1 is recognized as a positive NO carrier. The novel mutant enzymes E129K and Q109K are inferred to possess superior NO carrying capacity.
人体谷胱甘肽 S-转移酶（hGSTs）是Ⅱ相解毒酶，可催化亲电化合物与谷胱甘肽的结合。在疾病或应激状态下，细胞内环境中超量产生的一氧化氮（NO）会对细胞产生致命影响。研究表明，细胞进化出基于酪氨酸的 GSTs 作为一种防御机制，以减轻一氧化氮（NO）的毒性。hGSTP1 作为 NO 载体和清除剂的双重作用是随后研究的前奏。
考虑 hGSTM1 作为 NO 载体的可能作用。NO 作为一种重要的细胞信使和次级代谢物，过量产生对细胞是致命的。此外，hGSTM1 多态性导致催化活性降低，从而促进疾病状态。因此，生成比野生型（WT）具有更高催化效力的 hGSTM1 突变体是很有必要的。
使用计算机模拟和体外定点突变（SDM）生成具有更高效率的 hGSTM1 突变体。使用亲和层析法过表达和纯化 WT 和突变蛋白。测定 WT 和突变蛋白对 CDNB（1-氯-2,4-二硝基苯）和 NO 的催化活性和结合效率。
NO 测定表明 WT hGSTM1 与 NO 可能存在相互作用。计算机模拟、SDM 研究表明，E129K 和 Q109K 突变体比 WT 具有更高的 NO 结合效率。突变体的催化活性（GST 和 NO 测定）与计算机模拟结果相符。
WT hGSTM1 被认为是一种正向的 NO 载体。新型突变酶 E129K 和 Q109K 被推断具有更高的携带 NO 的能力。
