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Applied Biochemistry and Biotechnology

, Volume 183, Issue 3, pp 699–711 | Cite as

Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from Anabaena variabilis by Modification of Center-Near Surface Residues

  • Fan Zhang
  • Nan Huang
  • Li Zhou
  • Wenjing Cui
  • Zhongmei Liu
  • Longbao Zhu
  • Yi Liu
  • Zhemin ZhouEmail author
Article

Abstract

Phenylalanine ammonia lyase from Anabaena variabilis (Av-PAL) is a candidate for the treatment of phenylketonuria (PKU). However, Av-PAL shows its optimal pH at 8.5 and maintains only 70% of its highest activity when pH decreases to 7.3–7.4 (the condition of human plasma). The objective of the study was to shift its optimal pH by mutating surface amino acid residues which interact with the general base Tyr78. Based on the crystal structure and the online program GETAREA, we selected five sites: Asn69, Glu72, Glu75, Asn89, and Val90. Removing negative charges or introducing positive charges near the general base Tyr78 by mutation, the pH optima were successfully shifted to more acidic range. Especially, the pH optima of E75A, E75L, and E75Q were shifted to 7.5 with 35, 30, and 24% higher specific activities than that of the wild, respectively. Half-lives of E75L and E75Q at 70 °C prolonged to 190 and 180 min from 130 min of the wild, respectively. In addition, the higher resistance to a low pH of 3.5 and protease made E75L a candidate for oral medicine of PKU. This work would improve the therapeutic prospect of Av-PAL and provide guidance in modulating optimal pH of enzymes.

Keywords

Phenylalanine ammonia lyase pH shift Therapeutic application Mutation Surface residues Kinetic stability Resistance 

Notes

Acknowledgements

This work was mainly supported by Key Laboratory of Industrial Biotechnology, Ministry of Education, China.

Compliance with Ethical Standards

Funding Sources

This work was partly funded by the National Natural Science Foundation of China (31300087, 31400058, 31671797, and 21506172), the Natural Science Foundation of Jiangsu Province of China (BK20130131, BK20130139, and BK20140151), the National High Technology Research and Development Program of China (863 Program, 2014AA021304), the High Foreign Experts Project (GDW20123200114), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (111-2-06), the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program and the Fundamental Research Funds for the Central Universities (JUSRP51411B, JUSRP51504, JUSRP51611A), and the Natural Science Foundation of Anhui Province University of China (KJ2016A801).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12010_2017_2458_MOESM1_ESM.docx (164 kb)
ESM 1 (DOCX 164 kb)

Reference

  1. 1.
    Giberti, S., Bertea, C. M., Narayana, R., Maffei, M. E., & Forlani, G. (2012). Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae. Journal of Plant Physiology, 169, 249–254.CrossRefGoogle Scholar
  2. 2.
    Hou, X., Shao, F., Ma, Y., & Lu, S. (2013). The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis. Molecular Biology Reports, 40, 4301–4310.CrossRefGoogle Scholar
  3. 3.
    Jin, Q., Yao, Y., Cai, Y., & Lin, Y. (2013). Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from Dendrobium. PloS One, 8, e62352.CrossRefGoogle Scholar
  4. 4.
    Shang, Q. M., Li, L., & Dong, C. J. (2012). Multiple tandem duplication of the phenylalanine ammonia lyase genes in Cucumis. sativus L. Planta, 236, 1093–1105.CrossRefGoogle Scholar
  5. 5.
    Wang, X. H., Gong, M., Tang, L., Zheng, S., Lou, J. D., Ou, L., Gomes-Laranjo, J., & Zhang, C. (2013). Cloning, bioinformatics and the enzyme activity analyses of a phenylalanine ammonia-lyase gene involved in dragon’s blood biosynthesis in Dracaena cambodiana. Molecular Biology Reports, 40, 97–107.CrossRefGoogle Scholar
  6. 6.
    Gilbert, H. J., Clarke, I. N., Gibson, R. K., Stephenson, J. R., & Tully, M. (1985). Molecular cloning of the phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12. Journal of Bacteriology, 161, 314–320.Google Scholar
  7. 7.
    Zhu, L. B., Cui, W. J., Fang, Y. Q., Liu, Y., Gao, X. X., & Zhou, Z. M. (2013). Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis. Biotechnology Letters, 35, 751–756.CrossRefGoogle Scholar
  8. 8.
    Kim, M., Yoon, H., You, Y. H., Kim, Y. E., Woo, J. R., Seo, Y., Lee, G. M., Kim, Y. J., Kong, W. S., & Kim, J. G. (2013). Metagenomic analysis of fungal communities inhabiting the fairy ring zone of Tricholoma matsutake. Journal of Microbiology and Biotechnology, 23, 1347–1356.CrossRefGoogle Scholar
  9. 9.
    Vaslet, C. A., Strausberg, R. L., Sykes, A., Levy, A., & Filpula, D. (1988). cDNA and genomic cloning of yeast phenylalanine ammonia-lyase genes reveal genomic intron deletions. Nucleic Acids Research, 16, 11382.CrossRefGoogle Scholar
  10. 10.
    Moffitt, M. C., Louie, G. V., Bowman, M. E., Pence, J., Noel, J. P., & Moore, B. S. (2007). Discovery of two cyanobacterial phenylalanine ammonia lyases: kinetic and structural characterization. Biochemistry, 46, 1004–1012.CrossRefGoogle Scholar
  11. 11.
    Kovács, K., Bánóczi, G., Varga, A., Szabó, I., Holczinger, A., Hornyánszky, G., Zagyva, I., Paizs, C., Vértessy, B. G., & Poppe, L. (2014). Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus. PloS One, 9, e85943–e85943.CrossRefGoogle Scholar
  12. 12.
    Williams, J. S., Thomas, M., & Clarke, D. J. (2005). The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology, 151, 2543–2550.CrossRefGoogle Scholar
  13. 13.
    Fowler, Z. L., & Koffas, M. A. G. (2009). Biosynthesis and biotechnological production of flavanones: current state and perspectives. App. Microbiol. Biot., 83, 799–808.CrossRefGoogle Scholar
  14. 14.
    Horinouchi, S. (2009). Combinatorial biosynthesis of plant medicinal polyketides by microorganisms. Current Opinion in Chemical Biology, 13, 197–204.CrossRefGoogle Scholar
  15. 15.
    Kong, J. Q. (2015). Phenylalanine ammonia-lyase, a key component used for phenylpropanoids production by metabolic engineering. RSC Advances, 5, 62587–62603.Google Scholar
  16. 16.
    Babich, O. O., Pokrovsky, V. S., Anisimova, N. Y., Sokolov, N. N., & Prosekov, A. Y. (2013). Recombinant L-phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent. Biotechnol. Appl. Bioc., 60, 316–322.CrossRefGoogle Scholar
  17. 17.
    Shen, R. S., Fritz, R. R., & Abell, C. W. (1977). Clearance of phenylalanine ammonia-lyase from normal and tumor-bearing mice. Cancer Research, 37, 1051–1056.Google Scholar
  18. 18.
    Jaliani, H. Z., Farajnia, S., Mohammadi, S. A., Barzegar, A., & Talebi, S. (2013). Engineering and kinetic stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase. Applied Biochemistry and Biotechnology, 171, 1805–1818.CrossRefGoogle Scholar
  19. 19.
    Longo, N., Harding, C. O., Burton, B. K., Grange, D. K., Vockley, J., Wasserstein, M., Dorenbaum, A., Neuenburg, J. K., & Musson, D. G. (2014). Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial. Lancet, 384, 37–44.CrossRefGoogle Scholar
  20. 20.
    Sarkissian, C. N., Kang, T. S., Gámez, A., Scriver, C. R., & Stevens, R. C. (2011). Evaluation of orally administered PEGylated phenylalanine ammonia lyase in mice for the treatment of phenylketonuria. Molecular Genetics and Metabolism, 104, 249–254.CrossRefGoogle Scholar
  21. 21.
    Manikandan, K., Bhardwaj, A., Gupta, N., Lokanath, N. K., Ghosh, A., Reddy, V. S., & Ramakumar, S. (2006). Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: structural insights into alkalophilicity and implications for adaptation to polyextreme conditions. Protein Science, 15, 1951–1960.CrossRefGoogle Scholar
  22. 22.
    Cockburn, D. W., & Clarke, A. J. (2011). Modulating the pH-activity profile of cellulase A from Cellulomonas fimi by replacement of surface residues. Protein Engineering, Design & Selection, 24, 429–437.CrossRefGoogle Scholar
  23. 23.
    Tomschy, A., Brugger, R., Lehmann, M., Svendsen, A., Vogel, K., Kostrewa, D., Lassen, S. F., Burger, D., Kronenberger, A., van Loon, A. P. G. M., Pasamontes, L., & Wyss, M. (2002). Engineering of phytase for improved activity at low pH. Appl. Environ. Microb., 68, 1907–1913.CrossRefGoogle Scholar
  24. 24.
    Kim, T., Mullaney, E. J., Porres, J. M., Roneker, K. R., Crowe, S., Rice, S., Ko, T., Ullah, A. H. J., Daly, C. B., Welch, R., & Lei, X. G. (2006). Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. Appl. Environ. Microb., 72, 4397–4403.CrossRefGoogle Scholar
  25. 25.
    Hirata, A., Adachi, M., Utsumi, S., & Mikami, B. (2004). Engineering of the pH optimum of Bacillus cereus β-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type. Biochemistry, 43, 12523–12531.CrossRefGoogle Scholar
  26. 26.
    Cockburn, D. W., Vandenende, C., & Clarke, A. J. (2010). Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi. Biochemistry, 49, 2042–2050.CrossRefGoogle Scholar
  27. 27.
    Siddiqui, K. S., Lovinyanderton, T., Rangarajan, M., & Hartley, B. S. (1993). Arthrobacter D-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum. The Biochemical Journal, 296, 685–691.CrossRefGoogle Scholar
  28. 28.
    Cooke, H. A., Christianson, C. V., & Bruner, S. D. (2009). Structure and chemistry of 4-methylideneimidazole-5-one containing enzymes. Current Opinion in Chemical Biology, 13, 460–468.CrossRefGoogle Scholar
  29. 29.
    Fraczkiewicz, R., & Braun, W. (1998). Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. Journal of Computational Chemistry, 19, 319–333.CrossRefGoogle Scholar
  30. 30.
    Fiser, A., & Šali, A. (2003). Modeller: generation and refinement of homology-based protein structure models. Method. Enzymol., 374, 461–491.CrossRefGoogle Scholar
  31. 31.
    Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., & Karplus, M. (1983). CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. Journal of Computational Chemistry, 4, 187–217.CrossRefGoogle Scholar
  32. 32.
    Calabrese, J. C., Jordan, D. B., Boodhoo, A., Sariaslani, S., & Vannelli, T. (2004). Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Biochemistry, 43, 11403–11416.CrossRefGoogle Scholar
  33. 33.
    Pilbák, S., Tomin, A., Rétey, J., & Poppe, L. (2006). The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases. The FEBS Journal, 273, 1004–1019.CrossRefGoogle Scholar
  34. 34.
    Ostanin, K., Harms, E. H., Stevis, P. E., Kuciel, R., Zhou, M. M., & Van Etten, R. L. (1992). Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. J. Bio. Chem., 267, 22830–22836.Google Scholar
  35. 35.
    Kang, T. S., Wang, L., Sarkissian, C. N., Gamez, A., Scriver, C. R., & Stevens, R. C. (2010). Converting an injectable protein therapeutic into an oral form: phenylalanine ammonia lyase for phenylketonuria. Molecular Genetics and Metabolism, 99, 4–9.CrossRefGoogle Scholar
  36. 36.
    Jr, K. J., Nyberg, K., Sali, D., & Fersht, A. R. (2011). Contribution of hydrophobic interactions to protein stability. Journal of Molecular Biology, 408, 514–528.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Fan Zhang
    • 1
  • Nan Huang
    • 1
  • Li Zhou
    • 1
  • Wenjing Cui
    • 1
  • Zhongmei Liu
    • 1
  • Longbao Zhu
    • 2
  • Yi Liu
    • 3
  • Zhemin Zhou
    • 1
    Email author
  1. 1.Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  2. 2.School of Biological and Chemical EngineeringAnhui Polytechnic UniversityWuhuChina
  3. 3.Key Laboratory of Food and Biotechnology, School of Food and BiotechnologyXihua UniversityChengduChina

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