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Single-Point Mutation Near Active Center Increases Substrate Affinity of Alginate Lyase AlgL-CD

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Abstract

Alginate lyases have been widely used for the preparation of bioactive alginate oligosaccharides. An alginate lyase AlgL-CD was rationally designed by introducing alkaline amino acid residues near active center to increase activity. One of its mutants E226K presented much higher activity than wild-type AlgL-CD. Substrate affinity of E226K increased 10 folds as the Km values indicated. The spectra of intrinsic emission fluorescence and circular dichroism of E226K suggested the whole enzyme turned to be more flexible. The 8-anilino-1-naphthalenesulfonate (ANS)-binding assay showed that the hydrophobic active center of E226K was more available to ligand. Molecular dynamic analysis of the enzyme-substrate complex showed that lid loops of the active center in E226K turned to be more opened up, which might contribute to the increase of substrate-binding affinity. Meanwhile, the catalytic residue of E226K was closer to the hydrogen donor C5 atom of the substrate to increase catalysis rate. The final degradation products of alginate by E226K were determined to be identical with that of AlgL-CD. This study provides guidance for improving enzymatic preparation efficiency of bioactive alginate oligosaccharides.

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Data Availability

The datasets in this work are available from the corresponding author upon reasonable request.

References

  1. Wang, D. M., Aarstad, O. A., Li, J., McKee, L. S., Saetrom, G. I., Vyas, A., Srivastava, V., Aachmann, F. L., Bulone, V., & Hsieh, Y. S. Y. (2018). Preparation of 4-Deoxy-L-erythro-5-hexoseulose uronic acid (DEH) and guluronic acid rich alginate using a unique exo-alginate lyase from Thalassotalea crassostreae. Journal of Agricultural and Food Chemistry, 66(6), 1435–1443.

    Article  CAS  Google Scholar 

  2. Yamasaki, Y., Yokose, T., Nishikawa, T., Kim, D. Y., Jiang, Z. D., Yamaguchi, K., & Oda, T. (2012). Effects of alginate oligosaccharide mixtures on the growth and fatty acid composition of the green alga Chlamydomonas reinhardtii. Journal of Bioscience and Bioengineering, 113(1), 112–116.

    Article  CAS  Google Scholar 

  3. Bi, D., Lai, Q., Cai, N., Li, T., Zhang, Y., Han, Q., Peng, Y., Xu, H., Lu, J., Bao, W., Liu, Q., & Xu, X. (2018). Elucidation of the molecular-mechanisms and in vivo evaluation of the anti-inflammatory effect of alginate-derived seleno-polymannuronate. Journal of Agricultural and Food Chemistry, 66(9), 2083–2091.

    Article  CAS  Google Scholar 

  4. Zhou, R., Shi, X. Y., Gao, Y., Cai, N., Jiang, Z. D., & Xu, X. (2015). Anti-inflammatory activity of gluronate oligosaccharides obtained by oxidative degradation from alginate in lipopolysaccharide-activated murine macrophage RAW 264.7 cells. Journal of Agricultural and Food Chemistry, 63(1), 160–168.

    Article  CAS  Google Scholar 

  5. Tran, V. C., Cho, S. Y., Kwon, J., & Kim, D. (2019). Alginate oligosaccharide (AOS) improves immuno-metabolic systems by inhibiting STOML2 overexpression in high-fat-diet-induced obese zebrafish. Food & Function, 10(8), 4636–4648.

    Article  CAS  Google Scholar 

  6. Wang, Y., Song, Q. H., & Zhang, X. H. (2016). Marine microbiological enzymes: studies with multiple strategies and prospects, Mar. Drugs, 14, 171.

    Google Scholar 

  7. Wong, T. Y., Preston, L. A., & Schiller, N. L. (2000). Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annual Review of Microbiology, 54(1), 289–340.

    Article  CAS  Google Scholar 

  8. Xu, F., Wang, P., Zhang, Y. Z., & Chen, X. L. (2018). Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases. Applied and Environmental Microbiology, 84, 1–12.

    Google Scholar 

  9. Gurpilhares, D. B., Cinelli, L. P., Simas, N. K., Pessoa, A., & Setter Jr., L. D. (2019). Marine prebiotics: polysaccharides and oligosaccharides obtained by using microbial enzymes. Food Chemistry, 280, 175–186.

    Article  CAS  Google Scholar 

  10. Lyu, Q. Q., Zhang, K. K., Shi, Y. H., Li, W. H., Diao, X. T., & Liu, W. Z. (2019). Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution. Biochimica et Biophysica Acta-General Subjects, 1863(7), 1167–1176.

    Article  CAS  Google Scholar 

  11. Xu, F., Dong, F., Wang, P., Cao, H. Y., Li, C. Y., Li, P. Y., Pang, X. H., Zhang, Y. Z., & Chen, X. L. (2017). Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase family 6. The Journal of Biological Chemistry, 292(11), 4457–4468.

    Article  CAS  Google Scholar 

  12. Mikami, B., Ban, M., Suzuki, S., Yoon, H. J., Miyake, O., Yamasaki, M., Ogura, K., Maruyama, Y., Hashimoto, W., & Murata, K. (2012). Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III. Acta Crystallographica Section D, 68(9), 1207–1216.

    Article  CAS  Google Scholar 

  13. Dong, S., Wei, T. D., Chen, X. L., Li, C. Y., Wang, P., Xie, B. B., Qin, Q. L., Zhang, X. Y., Pang, X. H., Zhou, B. C., & Zhang, Y. Z. (2014). Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18. The Journal of Biological Chemistry, 289(43), 29558–29569.

    Article  CAS  Google Scholar 

  14. Han, W., Gu, J., Cheng, Y., Liu, H., Li, Y., & Li, F. (2016). Novel alginate lyase (Aly5) from a polysaccharide-degrading marine bacterium, Flammeovirga sp. strain MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties. Applied and Environmental Microbiology, 82(1), 364–374.

    Article  CAS  Google Scholar 

  15. Wang, B., Ji, S. Q., Ma, X. Q., Lu, M., Wang, L. S., & Li, F. L. (2018). Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase. FEBS Letters, 592(3), 369–379.

    Article  CAS  Google Scholar 

  16. Ogura, K., Yamasaki, M., Mikami, B., Hashimoto, W., & Murata, K. (2008). Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1. Journal of Molecular Biology, 380(2), 373–385.

    Article  CAS  Google Scholar 

  17. Yang, M., Yang, S. X., Liu, Z. M., Li, N. N., Li, L., & Mou, H. J. (2019). Rational design of alginate lyase from Microbulbifer sp. Q7 to improve thermal Stability. Marine Drugs, 17, 1–13.

    Google Scholar 

  18. Hammer, S. C., Knight, A. M., & Arnold, F. H. (2017). Design and evolution of enzymes for non-natural chemistry. Current Opinion in Green and Sustainable Chemistry, 7, 23–30.

    Article  Google Scholar 

  19. Han, W., Xu, X. Q., Ye, X. Y., & Lin, J. (2018). Purification and characterization of the alginate lyase isolated from marine. Journal of Fuzhou University (Natural Science Edition), 46, 136–142.

    Google Scholar 

  20. Reetz, M. T., Kahakeaw, D., & Lohmer, R. (2008). Addressing the numbers problem in directed evolution. ChemBioChem, 9(11), 1797–1804.

    Article  CAS  Google Scholar 

  21. Walker, J. M. (1994). The Bicinchoninic Acid (BCA) Assay for protein quantitation. In J. M. Walker (Ed.), Basic protein and peptide protocols (pp. 5–8). Totowa: Humana Press.

    Chapter  Google Scholar 

  22. Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S., & Olson, A. J. (2009). AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. Journal of Computational Chemistry, 30(16), 2785–2791.

    Article  CAS  Google Scholar 

  23. Case, D.A., Cerutti, D.S., Cheatham, T.E., Cruzeiro III, V.W.D., Darden, T.A., Duke, R.E., Ghoreishi, H.G., Goetz, A.W., Greene, D., Harris, R., and et al. (2018). AMBER 2018, University of California, San Francisco, (http://ambermd.org/doc12/Amber18.pdf)

  24. Li, Q., Hu, F., Zhu, B., Sun, Y., & Yao, Z. (2019). Biochemical characterization and elucidation of action pattern of a novel polysaccharide lyase 6 family alginate lyase from marine bacterium Flammeovirga sp. NJ-04, Mar. Drugs, 17, 1–11.

    Google Scholar 

  25. Hu, T., Li, C. X., Zhao, X., Li, G. S., Yu, G. L., & Guan, H. S. (2013). Preparation and characterization of guluronic acid oligosaccharides degraded by a rapid microwave irradiation method. Carbohydrate Research, 373, 53–58.

    Article  CAS  Google Scholar 

  26. Štěpán, T., Jan, K., Pavel, S., Samuli Ollila, O. H., & Pavel, J. (2018). Calcium sensing by recovering: effect of protein conformation on ion affinity. Journal of Physical Chemistry Letters, 9, 1613–1619.

    Article  Google Scholar 

  27. Belik, A. A., Silchenko, A. S., Kusaykin, M. I., Zvyagintseva, T. N., & Ermakova, S. P. (2018). Alginate lyases: substrates, structure, properties, and prospects of application. Russian Journal of Bioorganic Chemistry, 44(4), 386–396.

    Article  CAS  Google Scholar 

  28. Fischer, A., & Wefers, D. (2019). Chromatographic analysis of alginate degradation by five recombinant alginate lyases from Cellulophaga algicola DSM 14237. Food Chemistry, 299, 125142.

    Article  CAS  Google Scholar 

  29. Wong, S., Eckersley, E. L., Berger, B., & Klauda, J. B. (2019). Probing the pH effects on sugar binding to a polysaccharide lyase. The Journal of Physical Chemistry. B, 123(33), 7123–7136.

    Article  CAS  Google Scholar 

  30. Hong, S. Y., & Yoo, Y. J. (2013). Activity enhancement of Candida antarctica lipase B by flexibility modulation in helix region surrounding the active site. Applied Biochemistry and Biotechnology, 170(4), 925–933.

    Article  CAS  Google Scholar 

  31. Koshland, D. E. (1958). Application of a Theory of Enzyme Specificity to Protein Synthesis. Proceedings of the National Academy of Sciences of the United States of America, 44(2), 98–104.

    Article  CAS  Google Scholar 

  32. Tsou, C. L. (1988). Kinetics of substrate reaction during irreversible modification of enzyme activity. Advances in Enzymology and Related Areas of Molecular Biology, 61, 381–436.

    CAS  PubMed  Google Scholar 

  33. Sun, Z. T., Liu, Q., Qu, G., Feng, Y., & Reetz, M. T. (2019). Utility of B-factors in protein science: interpreting rigidity, flexibility, and internal motion and engineering thermostability. Chemical Reviews, 119(3), 1626–1665.

    Article  CAS  Google Scholar 

  34. Richard, J. P. (2019). Protein flexibility and stiffness enable efficient enzymatic catalysis. Journal of the American Chemical Society, 141(8), 3320–3331.

    Article  CAS  Google Scholar 

  35. Kim, H. T., Chung, J. H., Wang, D., Lee, J., Woo, H. C., Choi, I. G., & Kim, K. H. (2012). Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40. Applied Microbiology and Biotechnology, 93(5), 2233–2239.

    Article  CAS  Google Scholar 

  36. Lee, S. I., Choi, S. H., Lee, E. Y., & Kim, H. S. (2012). Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2. Applied Microbiology and Biotechnology, 95(6), 1643–1653.

    Article  CAS  Google Scholar 

  37. Xu, F., Chen, X. L., Sun, X. H., Dong, F., Li, C. Y., Li, P. Y., Ding, H. T., Chen, Y., Zhang, Y. Z., & Wang, P. (2020). Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the Arctic. Journal of Biological Chemistry, 295(48), 16380–16392. https://doi.org/10.1074/jbc.RA120.015106.

    Article  CAS  Google Scholar 

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Funding

This study was funded by Regional Development Project of Fujian Province (2019N3001).

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Authors and Affiliations

  1. College of Biological Sciences and Engineering, Fuzhou University, Fuzhou, 350108, China

    Xinqi Xu, Deyang Zeng, Dongyan Wu & Juan Lin

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  1. Xinqi Xu
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  2. Deyang Zeng
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  3. Dongyan Wu
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  4. Juan Lin
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Contributions

XQX: investigation, writing—original draft preparation; DYZ: investigation; DYW: investigation; JL: supervision, conceptualization, and methodology.

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Correspondence to Juan Lin.

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Xu, X., Zeng, D., Wu, D. et al. Single-Point Mutation Near Active Center Increases Substrate Affinity of Alginate Lyase AlgL-CD. Appl Biochem Biotechnol 193, 1513–1531 (2021). https://doi.org/10.1007/s12010-021-03507-x

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