Background: Ochratoxin A (OTA), a hazardous mycotoxin, poses significant health risks through food contamination. Developing efficient enzymatic degradation strategies is crucial for food safety. This study investigates the catalytic mechanism of ADH3 amidohydrolase in OTA detoxification using multiscale computational approaches. Methods and Results: Bioinformatics analysis revealed ADH3’s conserved catalytic motifs and critical residues, including the zinc-coordination network and catalytic gatekeeper Asp344. Structural modeling characterized the enzyme’s hydrophobic core and substrate-binding pocket. Site-directed mutagenesis and molecular docking simulations demonstrated that OTA binding is stabilized by key hydrophobic and electrostatic interactions. Molecular dynamics simulations further elucidated the dynamic behavior of the enzyme-substrate complex, highlighting the role of conformational flexibility in catalysis. Conclusion: This study provides mechanistic insights into ADH3’s gatekeeping function, establishing a computational framework for rational design of high-efficiency enzymes against mycotoxin contamination.
References
[1] Zhang, Z., Qi, X., Wang, Y., Li, Y., Cheng, L., Yang, Q., Luo, J. (2025) Fungi and mycotoxin types, toxicity, mechanisms, and control of mycotoxin contamination: a review. Food Additives Contaminants: Part A, 42(12), 1692-1716.
[2] Wang, L., Hua, X., Shi, J., Jing, N., Ji, T., Lv, B., Liu, L., Chen, Y. (2022) Ochratoxin A: occurrence and recent advances in detoxification. Toxicon, 210, 11-18.
[3] Bostan, H. B., Danesh, N. M., Karimi, G., Ramezani, M., Shaegh, S. A. M., Youssefi, K., Charbgoo, F., Abnous, K., Taghdisi, S. M. (2017) Ultrasensitive detection of ochratoxin A using aptasensors. Biosensors and Bioelectronics, 98, 168-179.
[4] Stoev, S. D. (2021) Follow up long term preliminary studies on carcinogenic and toxic effects of ochratoxin A in rats and the putative protection of phenylalanine. Toxicon, 190, 41-49.
[5] Stoev, S. D. (2020) Long term preliminary studies on toxic and carcinogenic effects of individual or simultaneous exposure to ochratoxin A and penicillic acid in mice. Toxicon, 184, 192-201.
[6] Yu, J., Smith, I. N., Mikiashvili, N. (2020) Reducing ochratoxin A content in grape pomace by different methods. Toxins, 12(7), 424.
[7] Huang, D., Zhang, L., Sun, Y. (2023) Rational design of disulfide bridges in Bb PETaseCD for enhancing the enzymatic performance in PET degradation. Molecules, 28(8), 3528.
[8] Chen, L., Li, Q., Nasif, K. F. A., Xie, Y., Deng, B., Niu, S., Pouriyeh S, Dai Z, Chen J, Xie, C. Y. (2024) AI-driven deep learning techniques in protein structure prediction. International Journal of Molecular Sciences, 25(15), 8426.
[9] Wu, X., Xu, L. Y., Li, E. M., Dong, G. (2022) Application of molecular dynamics simulation in biomedicine. Chemical Biology & Drug Design, 99(5), 789-800.
[10] de Oliveira, C., Leswing, K., Feng, S., Kanters, R., Abel, R., Bhat, S. (2023) FEP protocol builder: optimization of free energy perturbation protocols using active learning. Journal of Chemical Information and Modeling, 63(17), 5592-5603.
[11] Cao, L., Caldararu, O., Ryde, U. (2018) Protonation and reduction of the FeMo cluster in nitrogenase studied by quantum mechanics/molecular mechanics (QM/MM) calculations. Journal of Chemical Theory and Computation, 14(12), 6653-6678.
[12] Goodsell, D. S., Burley, S. K. (2020) RCSB protein data bank tools for 3D structure-guided cancer research: Human papillomavirus (HPV) case study. Oncogene, 39(43), 6623-6632.
[13] Rosignoli, S., Paiardini, A. (2022) Boosting the full potential of PyMOL with structural biology plugins. Biomolecules, 12(12), 1764.
[14] O’Connell, K. A., Yosufzai, Z. B., Campbell, R. A., Lobb, C. J., Engelken, H. T., Gorrell, L. M., Carlson, T. B., Catana, J. J., Mikdadi, D, Bonazzi VR, Klenk, J. A. (2023) Accelerating genomic workflows using NVIDIA Parabricks. BMC Bioinformatics, 24(1), 221.
[15] Osipov, E. M., Strelkov, S. V. (2025) DDtrek: A PyMOL-based management system for 3D structural data series. ACS Omega, 10(21), 21024-21029.
[16] Yang, Z., Zeng, X., Zhao, Y., Chen, R. (2023) AlphaFold2 and its applications in the fields of biology and medicine. Signal Transduction and Targeted Therapy, 8(1), 115.
[17] Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., de Beer. T.A.P., Rempfer. C., Bordoli. L., Lepore. R., Schwede, T. (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Research, 46(W1), W296-W303.
[18] Lee, J. W., Won, J. H., Jeon, S., Choo, Y., Yeon, Y., Oh, J. S., Kim, M., Kim, S., Joung, I., Jang, C., Lee, S. J., Kim, T. H., Jin, K. H., Song, G., Kim, E. S., Yoo, J., Paek, E., Noh, Y. K., Joo, K. (2023) DeepFold: Enhancing protein structure prediction through optimized loss functions, improved template features, and re-optimized energy function. Bioinformatics, 39(12), btad712.
[19] Vatanpour, H., Najafi, M., Ahmadinejad, M., Bayatzadeh, M. A. (2023) Biochemistry of the thrombin-like enzyme and its purification from Iranian Echis carinatus snake venom: its interaction with platelet receptors. Archives of Razi Institute, 78(6), 1822.
[20] Petratos, K., Gessmann, R., Daskalakis, V., Papadovasilaki, M., Papanikolau, Y., Tsigos, I., Bouriotis, V. (2020) Structure and dynamics of a thermostable alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123. ACS Omega, 5(24), 14523-14534.
[21] Khammar, N., Heidari, M. M., Khatami, M., Shahshahani, R. (2024) Mutational analysis and prediction of the potential impact of missense mutations in the HOXA9 gene in B-cell acute lymphoblastic leukemia. Human Gene, 41, 201318.
[22] Kanapeckaite, A. (2021) Fiscore package: effective protein structural data visualisation and exploration. Artificial Intelligence in the Life Sciences, 1, 100016.
[23] Virk, N., Kumari, U. (2022) Genome sequence analysis of lungs cancer protein WDR74 (WD Repeat-Containing Protein). IJRASET 10 (V): 4533, 4537.
[24] Keklik, G. (2023) Understanding evolutionary relationships and analysis methods through mega software. International Journal of New Horizons in the Sciences, 83-90.
[25] Liu, Y., Yang, X., Gan, J., Chen, S., Xiao, Z. X., Cao, Y. (2022) CB-Dock2: improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Research, 50(W1), W159-W164.
[26] Paul, S. K., Saddam, M., Rahaman, K. A., Choi, J. G., Lee, S. S., Hasan, M. (2022) Molecular modeling, molecular dynamics simulation, and essential dynamics analysis of grancalcin: an upregulated biomarker in experimental autoimmune encephalomyelitis mice. Heliyon, 8(10).
[27] Smith, M. D., Rao, J. S., Segelken, E., Cruz, L. (2015) Force-field induced bias in the structure of Aβ21-30: a comparison of OPLS, AMBER, CHARMM, and GROMOS force fields. Journal of Chemical Information and Modeling, 55(12), 2587-2595.
[28] Zhao, M., Kim, J., Guo, C., Jang, J. (2025) Distinct mechanisms for the underwater adhesions of Pvfp-5β and Mfp-5: a comparative all-atom molecular dynamics study. Surfaces and Interfaces, 58, 105847.
[29] Yang, P., Zheng, W., Ning, K., Zhang, Y. (2021) Decoding the link of microbiome niches with homologous sequences enables accurately targeted protein structure prediction. Proceedings of the National Academy of Sciences, 118(49), e2110828118.
[30] Guo, X., Zhou, S., Wang, Y., Song, J., Wang, H., Kong, D., Zhu, J., Dong, W., He, M., Hu, G., Ruan, Z. (2016) Characterization of a highly thermostable and organic solvent-tolerant copper-containing polyphenol oxidase with dye-decolorizing ability from Kurthia huakuii LAM0618T. PLOS One, 11(10), e0164810.
[31] Wu, Z. M., Zheng, R. C., Zheng, Y. G. (2017) Identification and characterization of a novel amidase signature family amidase from Parvibaculum lavamentivorans ZJB14001. Protein Expression and Purification, 129, 60-68.
[32] Hilton, J., Hall, I. (2024) A beta-Poisson model for infectious disease transmission. PLoS Computational Biology, 20(2), e1011856.
[33] Zhang, M., Wang, S., Xu, R., Liu, Y., Zhang, H., Sun, M., Wang, J., Liu, Z., Wu, K. (2023) Managing genomic diversity in conservation programs of Chinese domestic chickens. Genetics Selection Evolution, 55(1), 92.
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Lu, S., Du, J., Ren, X., Yan, M., Wang, S., Yang, Z., Yang, X., Zhang, Y., Min, X., Hua, C., Zhao, Y., Zhang, R., Shen, Z., Nie, S., Tian, J., Zhang, N., Liao, W. (2025) Multiscale Computational Elucidation of the Catalytic Gatekeeping Mechanism in ADH3 Amidohydrolase for Ochratoxin a Detoxification. Scientific Research Bulletin, 2(4), 53-67. https://doi.org/10.71052/srb2024/FTFI8215