Heuristics in Role of Human Glutathione S-transferase Mu 1 as Nitric Oxide Carrier and its Engineered Variants for...

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.