Glutathione (GSH) is a tripeptide compound that contains an γ-amide bond and a sulfhydryl group.It is widely used in the fields of medicine, food, and cosmetics.The biosynthesis of glutathione involves two main steps, including γ-glutamylcysteine synthetase (GSHA) and glutathione synthetase (GSHB).The production of glutathione using natural enzymes was found to be inefficient due to the rate-limiting enzyme GSHA being poorly catalysed and inhibited by feedback from the product.This study analysed GSHA from Escherichia coli, molecularly modified it, and identified the active sites involved in the transformation of GSHA, making key amino acid substitutions through alanine scanning.This study analysed amino acids located in inactive regions on the protein surface and introduced negatively charged amino acids to improve stability.The Y131A-A511D mutant with higher catalytic activity was obtained by combination of mutation sites with increased enzyme activity, which exhibited a higher catalytic effect and a 6-fold increase in enzyme activity compared to the wild type.The enzymatic properties of the best mutant were analysed.The mutant Y131A-A511D was used to catalyse the synthesis of glutathione, resulting in the production of 76.8 mmol/L of glutathione within 8 hours.This demonstrates efficient the catalytic production of glutathione.
[1] KENDALL E C, MCKENZIE B F, MASON H L.A study of glutathione[J].Journal of Biological Chemistry, 1929, 84(2):657-674.
[2] JONES D P.Redox potential of GSH/GSSG couple:Assay and biological significance[J].Methods in Enzymology, 2002, 348:93-112.
[3] PENNINCKX M J.An overview on glutathione in Saccharomyces versus non-conventional yeasts[J].FEMS Yeast Research, 2002, 2(3):295-305.
[4] CARMEL-HAREL O, STORZ G.Roles of the glutathione and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress[J].Annual Review of Microbiology, 2000, 54(1):439-461.
[5] HOMMA T, FUJII J.Application of glutathione as anti-oxidative and anti-aging drugs[J].Current Drug Metabolism, 2015, 16(7):560-571.
[6] FANG Y Z, YANG S, WU G.Free radicals, antioxidants, and nutrition[J].Nutrition, 2002, 18(10):872-879.
[7] LEI X G.In vivo antioxidant role of glutathione peroxidase:Evidence from knockout mice[J].Methods in Enzymology, 2002, 347:213-225.
[8] WU G Y, LUPTON J R, TURNER N D, et al.Glutathione metabolism and its implications for health[J].The Journal of Nutrition, 2004, 134(3):489-492.
[9] NIU B Y, LIAO K X, ZHOU Y X, et al.Application of glutathione depletion in cancer therapy:Enhanced ROS-based therapy, ferroptosis, and chemotherapy[J].Biomaterials, 2021, 277:121110.
[10] TOWNSEND D M, TEW K D, TAPIERO H.The importance of glutathione in human disease[J].Biomedicine & Pharmacotherapy, 2003, 57(3-4):145-155.
[11] GUAN X M. Glutathione and glutathione disulfide - their biomedical and pharmaceutical applications[J]. Medicinal Chemistry Research, 2023, 32(9): 1972-1994.
[12] HARA K Y, KONDO A.ATP regulation in bioproduction[J].Microbial Cell Factories, 2015, 14:198.
[13] ZHANG J, QUAN C, WANG C, et al.Systematic manipulation of glutathione metabolism in Escherichia coli for improved glutathione production[J].Microbial Cell Factories, 2016, 15(1):38.
[14] LU S C.Regulation of glutathione synthesis[J].Current Topics in Cellular Regulation, 2000, 36:95-116.
[15] RICHMAN P G, MEISTER A.Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione[J].Journal of Biological Chemistry, 1975, 250(4):1422-1426.
[16] MURATA K, KIMURA A.Overproduction of glutathione and its derivatives by genetically engineered microbial cells[J].Biotechnology Advances, 1990, 8(1):59-96.
[17] BITEROVA E I, BARYCKI J J.Mechanistic details of glutathione biosynthesis revealed by crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase[J].Journal of Biological Chemistry, 2009, 284(47):32 700-32 708.
[18] CHOI J M, KIM H S.Structure-guided rational design of the substrate specificity and catalytic activity of an enzyme[J].Methods in Enzymology, 2020, 643:181-202.
[19] ANAND P, NAGARAJAN D, MUKHERJEE S, et al.ABS-Scan:In silico alanine scanning mutagenesis for binding site residues in protein-ligand complex[J].F1000Research, 2014, 3:214.
[20] LIU X, PENG L, ZHOU Y F, et al.Computational alanine scanning with interaction entropy for protein–ligand binding free energies[J].Journal of Chemical Theory and Computation, 2018, 14(3):1772-1780.
[21] YANG J X, WANG B, GE L, et al.The enhancement of surface activity and nanoparticle stability through the alteration of charged amino acids of HGFI[J].Colloids and Surfaces B:Biointerfaces, 2019, 175:703-712.
[22] ANUMALLA B, PRABHU N P.Surface hydration and preferential interaction directs the charged amino acids-induced changes in protein stability[J].Journal of Molecular Graphics and Modelling, 2020, 98:107602.
[23] CUI C X, MING H, LI L J, et al.Fabrication of an in-situ co-immobilized enzyme in mesoporous silica for synthesizing GSH with ATP regeneration[J].Molecular Catalysis, 2020, 486:110870.
[24] 张浩月, 穆玉敏, 秦浩杰, 等.多酶体系中谷胱甘肽HPLC分析方法研究[J].煤炭与化工, 2023, 46(10):151-155.
ZHANG H Y, MU Y M, QIN H J, et al.Study on HPLC analysis method of glutathione in multienzyme system[J].Coal and Chemical Industry, 2023, 46(10):151-155.
[25] HIBI T, NII H, NAKATSU T, et al.Crystal structure of γ-glutamylcysteine synthetase:Insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis[J].Proceedings of the National Academy of Sciences, 2004, 101(42):15 052-15 057.
[26] SONG Z D, ZHANG Q F, WU W H, et al.Rational design of enzyme activity and enantioselectivity[J].Frontiers in Bioengineering and Biotechnology, 2023, 11:1129149.
[27] STEINER K, SCHWAB H.Recent advances in rational approaches for enzyme engineering[J].Computational and Structural Biotechnology Journal, 2012, 2(3):e201209010.
[28] LI W, LI Z M, YE Q.Enzymatic synthesis of glutathione using yeast cells in two-stage reaction[J].Bioprocess and Biosystems Engineering, 2010, 33(6):675-682.
[29] LI W, LI Z M, YANG J H, et al.Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli[J].Journal of Biotechnology, 2011, 154(4):261-268.
[30] JIANG Y, TAO R S, SHEN Z Q, et al.Enzymatic production of glutathione by bifunctional γ-glutamylcysteine synthetase/glutathione synthetase coupled with in vitro acetate kinase-based ATP generation[J].Applied Biochemistry and Biotechnology, 2016, 180(7):1446-1455.
