Heparin, a highly sulfated glycosaminoglycan, exhibits critical biological activities including anticoagulation, antilipidemia, anti-inflammation, antitumor effects, and bacterial adhesion inhibition, driving its substantial demand in medical and biotechnological applications.Aryl sulfotransferase IV (AST Ⅳ) plays a pivotal role in the microbial-enzymatic synthesis of heparin by enabling efficient synthesis and cost-effective regeneration of the sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS).This study optimized the codon usage of AST Ⅳ based on codon preference, constructed the recombinant expression vector pET32b-AST Ⅳ, and achieved its heterologous expression in Escherichia coli.Site-directed mutagenesis substituted cysteine residues with alanine, while molecular dynamics simulations elucidated the impact of disulfide bonds on enzymatic activity.Results demonstrated that mutations at Cys66 and Cys82 significantly reduced catalytic efficiency [kcat/Km values:(80.53±23.44), (104.85±25.17) L/(s·mol), respectively] compared to wild-type AST Ⅳ[(139.13±25.06) L/(s·mol)].AST Ⅳ Cys232 produced in Origami(DE3) exhibited 1.2-fold higher activity than that from BL21(DE3).Molecular simulations confirmed a disulfide bond between Cys66 and Cys82, with enhanced structural folding directly correlating to improved PAPS regeneration efficiency.These findings highlight the critical role of disulfide bond formation in modulating AST Ⅳ activity, providing insights for rational enzyme engineering to optimize heparin biosynthesis.
YUAN Shuming
,
LI Congxu
,
LI Chen
,
SHI Changqian
,
ZHOU Xianxuan
. The role of disulfide bonds in aryl sulfotransferase IV using site-directed mutagenesis[J]. Food and Fermentation Industries, 2025
, 51(23)
: 45
-52
.
DOI: 10.13995/j.cnki.11-1802/ts.042401
[1] QIU M, HUANG S J, LUO C H, et al.Pharmacological and clinical application of heparin progress:An essential drug for modern medicine[J].Biomedecine & Pharmacotherapie, 2021, 139:111561.
[2] HAO C, XU H M, YU L F, et al.Heparin:An essential drug for modern medicine[J].Progress in Molecular Biology and Translational Science, 2019, 163:1-19.
[3] WARDROP D, KEELING D.The story of the discovery of heparin and warfarin[J].British Journal of Haematology, 2008, 141(6):757-763.
[4] XU Y M, MASUKO S, TAKIEDDIN M, et al.Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins[J].Science, 2011, 334(6055):498-501.
[5] ZHANG X, XU Y M, HSIEH P H, et al.Chemoenzymatic synthesis of unmodified heparin oligosaccharides:Cleavage of p-nitrophenyl glucuronide by alkaline and Smith degradation[J].Organic & Biomolecular Chemistry, 2017, 15(5):1222-1227.
[6] YU Y Y, GONG B X, WANG H L, et al.Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan[J].Biotechnology and Bioengineering, 2023, 120(4):1081-1096.
[7] DATTA P, FU L, HE W Q, et al.Expression of enzymes for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis and their preparation for PAPS synthesis and regeneration[J].Applied Microbiology and Biotechnology, 2020, 104(16):7067-7078.
[8] AN C Y, ZHAO L, WEI Z J, et al.Chemoenzymatic synthesis of 3′-phosphoadenosine-5′-phosphosulfate coupling with an ATP regeneration system[J].Applied Microbiology and Biotechnology, 2017, 101(20):7535-7544.
[9] SCHIFF J A.Pathways of assimilatory sulphate reduction in plants and microorganisms[J].Ciba Foundation Symposium, 1979(72):49-69.
[10] GRIMSHAW J P A, STIRNIMANN C U, BROZZO M S, et al.DsbL and DsbI form a specific dithiol oxidase system for periplasmic arylsulfate sulfotransferase in uropathogenic Escherichia coli[J].Journal of Molecular Biology, 2008, 380(4):667-680.
[11] DATTA A K, CHAMMAS R, PAULSON J C.Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulfide bond[J].The Journal of Biological Chemistry, 2001, 276(18):15200-15207.
[12] HWA J, REEVES P J, KLEIN-SEETHARAMAN J, et al.Structure and function in rhodopsin:Further elucidation of the role of the intradiscal cysteines, cys-110, -185, and-187, in rhodopsin folding and function[J].Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(5):1932-1935.
[13] LEE J H, BLABER M.Structural basis of conserved cysteine in the fibroblast growth factor family:Evidence for a vestigial half-cystine[J].Journal of Molecular Biology, 2009, 393(1):128-139.
[14] HORWITZ A H, LEIGH S D, ABRAHAMSON S, et al.Expression and characterization of cysteine-modified variants of an amino-terminal fragment of bactericidal/permeability-increasing protein[J].Protein Expression and Purification, 1996, 8(1):28-40.
[15] GAO X, DONG X Q, LI X X, et al.Prediction of disulfide bond engineering sites using a machine learning method[J].Scientific Reports, 2020, 10:10330.
[16] BURKART M D, IZUMI M, WONG C H.Enzymatic regeneration of 3′-phosphoadenosine-5′-phosphosulfate using aryl sulfotransferase for the preparative enzymatic synthesis of sulfated carbohydrates[J].Angewandte Chemie (International Ed), 1999, 38(18):2747-2750.
[17] XU R R, ZHANG W, XI X T, et al.Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds[J].Nature Communications, 2023, 14:7297.
[18] BERGER I, GUTTMAN C, AMAR D, et al.The molecular basis for the broad substrate specificity of human sulfotransferase 1A1[J].PLoS One, 2011, 6(11):e26794.
[19] LU J H, LI H T, LIU M C, et al.Crystal structure of human sulfotransferase SULT1A3 in complex with dopamine and 3′-phosphoadenosine 5′-phosphate[J].Biochemical and Biophysical Research Communications, 2005, 335(2):417-423.
[20] COOK I, WANG T, ALMO S C, et al.The gate that governs sulfotransferase selectivity[J].Biochemistry, 2013, 52(2):415-424.
[21] ZHOU Z X, LI Q, XU R R, et al.Secretory expression of the rat aryl sulfotransferases IV with improved catalytic efficiency by molecular engineering[J].3 Biotech, 2019, 9(6):246.
