Food and Fermentation Industries >

2026 , Vol. 52 >Issue 7: 26 - 33

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.043702

Improving thermostability and substrate binding affinity of glutamate decarboxylase by integrating ProteinMPNN and molecular dynamics simulation

  • WANG Fuhao ,
  • ZHANG Wenwen ,
  • WU Xinming ,
  • LIU Fufeng
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  • (College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)

Received date: 2025-06-26

  Revised date: 2025-08-26

  Online published: 2026-04-17

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Abstract

With the rapid development of deep learning, protein design tools such as ProteinMPNN have emerged, enabling efficient enzyme performance optimization when combined with molecular dynamics simulations.In this study, glutamate decarboxylase (GAD) was selected as the target enzyme.Based on its evolutionary and structural information, 30 GAD variants (Mut1-Mut30) were designed using ProteinMPNN.The three-dimensional structures of these mutants were subsequently predicted using AlphaFold2, and MD simulations were performed at 310 K and 320 K.To evaluate structural stability, root mean square deviation analysis was performed, leading to the identification of a highly stable mutant, Mut12.To further investigate its thermostability and substrate-binding capacity, Mut12 was simulated for 300 ns at both 350 K and 310 K.The MD results demonstrated that Mut12 maintained good structural stability under high-temperature conditions (350 K), with a binding energy approximately 22% higher than that of the wild-type enzyme.Mechanistic analysis revealed that the D28P and K34P mutations enhanced the rigidity of the N-terminal region, which synergistically contributed to the formation of a substrate-binding pocket more complementary to the ligand.This study provides a novel strategy and theoretical foundation for the rational design and industrial application of high-performance enzymes.

Key words： rational design; glutamate decarboxylase; molecular dynamics simulation; ProteinMPNN; thermostability

Cite this article

WANG Fuhao , ZHANG Wenwen , WU Xinming , LIU Fufeng . Improving thermostability and substrate binding affinity of glutamate decarboxylase by integrating ProteinMPNN and molecular dynamics simulation[J]. Food and Fermentation Industries, 2026 , 52(7) : 26 -33 . DOI: 10.13995/j.cnki.11-1802/ts.043702

References

[1] COBB R E, CHAO R, ZHAO H M.Directed evolution:Past, present, and future[J].AIChE Journal, 2013, 59(5):1432-1440.
[2] ARNOLD F H.Directed evolution:Bringing new chemistry to life[J].Angewandte Chemie International Edition, 2018, 57(16):4143-4148.
[3] BEDNAR D, BEERENS K, SEBESTOVA E, et al.FireProt:Energy-and evolution-based computational design of thermostable multiple-point mutants[J].PLoS Computational Biology, 2015, 11(11):e1004556.
[4] GOLDENZWEIG A, GOLDSMITH M, HILL S E, et al.Automated structure-and sequence-based design of proteins for high bacterial expression and stability[J].Molecular Cell, 2016, 63(2):337-346.
[5] KHERSONSKY O, LIPSH R, AVIZEMER Z, et al.Automated design of efficient and functionally diverse enzyme repertoires[J].Molecular Cell, 2018, 72(1):178-186.e5.
[6] KAO H W, LU W L, HO M R, et al.Robust design of effective allosteric activators for Rsp5 E3 ligase using the machine learning tool ProteinMPNN[J].ACS Synthetic Biology, 2023, 12(8):2310-2319.
[7] HUANG Z L, NI D W, CHEN Z W, et al.Application of molecular dynamics simulation in the field of food enzymes:Improving the thermal-stability and catalytic ability[J].Critical Reviews in Food Science and Nutrition, 2024, 64(31):11396-11408.
[8] CHEN X H, CHEN Y J, TANG D D, et al.Recent advances in bioinspired multienzyme engineering for food applications[J].Trends in Food Science & Technology, 2025, 156:104840.
[9] LI J L, WANG S J, LIU C, et al.Going beyond the local catalytic activity space of chitinase using a simulation-based iterative saturation mutagenesis strategy[J].ACS Catalysis, 2022, 12(16):10235-10244.
[10] SUMIDA K H, NÚÑEZ-FRANCO R, KALVET I, et al.Improving protein expression, stability, and function with ProteinMPNN[J].Journal of the American Chemical Society, 2024, 146(3):2054-2061.
[11] ABBASS J, NEBEL J C.Rosetta and the journey to predict proteins’ structures, 20 years on[J].Current Bioinformatics, 2020, 15(6):611-628.
[12] RICHTER F, LEAVER-FAY A, KHARE S D, et al.De novo enzyme design using Rosetta3[J].PLoS One, 2011, 6(5):e19230.
[13] ZHU E H, XIANG X, WAN S D, et al.Discovery of the key mutation site influencing the thermostability of Thermomyces lanuginosus lipase by Rosetta design programs[J].International Journal of Molecular Sciences, 2022, 23(16):8963.
[14] LIU Z Y, GUO X L, DAI K Q, et al.Biosynthesis of gamma-aminobutyric acid by engineered Clostridium tyrobutyricum co-overexpressing glutamate decarboxylase and class I heat shock protein[J].Fermentation, 2023, 9(5):445.
[15] ZHU F, HU S, ZHAO W R, et al.A novel method for γ-aminobutyric acid biosynthesis using glutamate decarboxylase entrapped in polyvinyl alcohol-sodium alginate capsules[J].Molecules, 2023, 28(19):6844.
[16] UENO H.Enzymatic and structural aspects on glutamate decarboxylase[J].Journal of Molecular Catalysis B:Enzymatic, 2000, 10(1-3):67-79.
[17] FENALTI G, LAW R H P, BUCKLE A M, et al.GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop[J].Nature Structural & Molecular Biology, 2007, 14(4):280-286.
[18] LIU S J, WEN B T, DU G M, et al.Coordinated regulation of Bacteroides thetaiotaomicron glutamate decarboxylase activity by multiple elements under different pH[J].Food Chemistry, 2023, 403:134436.
[19] YAO L L, LYU C J, WANG Y T, et al.High-level production of γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli[J].Engineering Microbiology, 2023, 3(2):100077.
[20] 方卉, 吕常江, 花雨娇, 等.利用脯氨酸效应提高短乳杆菌谷氨酸脱羧酶的热稳定性[J].生物工程学报, 2019,35(4):636-646.
FANG H, LYU C J, HUA Y J, et al.Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline[].Chinese Journal of Biotechnology, 2019,35(4):636-646.
[21] CHA X C, DING J J, BA W Y, et al.High production of γ-aminobutyric acid by activating the xyl operon of Lactobacillus brevis[J].ACS Omega, 2023, 8(8):8101-8109.
[22] SHI F, XIE Y L, JIANG J J, et al.Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH[J].Enzyme and Microbial Technology, 2014, 61-62:35-43.
[23] YANG L J, ZHANG X, CHEN J, et al.Expanding the pH range of glutamate decarboxylase from L.pltarum LC84 by site-directed mutagenesis[J].Frontiers in Bioengineering and Biotechnology, 2023, 11:1160818.
[24] HUANG J, FANG H, GAI Z C, et al.Lactobacillus brevis CGMCC 1306 glutamate decarboxylase:Crystal structure and functional analysis[J].Biochemical and Biophysical Research Communications, 2018, 503(3):1703-1709.
[25] STEINEGGER M, MEIER M, MIRDITA M, et al.HH-suite3 for fast remote homology detection and deep protein annotation[J].BMC Bioinformatics, 2019, 20(1):473.
[26] GABLER F, NAM S Z, TILL S, et al.Protein sequence analysis using the MPI Bioinformatics Toolkit[J].Current Protocols in Bioinformatics, 2020, 72(1):e108.
[27] JOHN R A.Pyridoxal phosphate-dependent enzymes[J].Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1995, 1248(2):81-96.
[28] ABRAHAM M J, MURTOLA T, SCHULZ R, et al.GROMACS:High performance molecular simulations through multi-level parallelism from laptops to supercomputers[J].SoftwareX, 2015, 1-2:19-25.
[29] SCHMID N, EICHENBERGER A P, CHOUTKO A, et al.Definition and testing of the GROMOS force-field versions 54A7 and 54B7[J].European Biophysics Journal, 2011, 40(7):843-856.
[30] LU T.A comprehensive electron wavefunction analysis toolbox for chemists, Multiwfn[J].The Journal of Chemical Physics, 2024, 161(8).DOI:10.1063/5.0216272.
[31] MAIER J A, MARTINEZ C, KASAVAJHALA K, et al.Ff14SB:Improving the accuracy of protein side chain and backbone parameters from ff99SB[J].Journal of Chemical Theory and Computation, 2015, 11(8):3696-3713.
[32] OSVÁTH S, GRUEBELE M.Proline can have opposite effects on fast and slow protein folding phases[J].Biophysical Journal, 2003, 85(2):1215-1222.
[33] YANG X W, HUO X J, TANG Y Q, et al.Integrating enzyme evolution and metabolic engineering to improve the productivity of γ-aminobutyric acid by whole-cell biosynthesis in Escherichia coli[J].Journal of Agricultural and Food Chemistry, 2023, 71(11):4656-4664.
[34] VALDÉS-TRESANCO M S, VALDÉS-TRESANCO M E, VALIENTE P A, et al.Gmx_MMPBSA:A new tool to perform end-state free energy calculations with GROMACS[J].Journal of Chemical Theory and Computation, 2021, 17(10):6281-6291.
[35] BOUYSSET C, FIORUCCI S.ProLIF:A library to encode molecular interactions as fingerprints[J].Journal of Cheminformatics, 2021, 13(1):72.
[36] YE B W, TIAN W, WANG B S, et al.CASTpFold:Computed atlas of surface topography of the universe of protein folds[J].Nucleic Acids Research, 2024, 52(W1):W194-W199.
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