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Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins

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Abstract

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s−1. This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

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References

  1. Higgins, L. J., Yan, F., Liu, P., Liu, H. W., & Drennan, C. L. (2005). Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature, 437, 838–844.

    Article  CAS  Google Scholar 

  2. Laden, B. P., Tang, Y., & Porter, T. D. (2000). Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase. Archives of Biochemistry and Biophysics., 374, 381–388.

    Article  CAS  Google Scholar 

  3. Hieber, A. D., Bugos, R. C., & Yamamoto, H. Y. (2000). Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase. Biochimica et Biophysica Acta, 1482, 84–91.

    Article  CAS  Google Scholar 

  4. Hartmans, S., van der Werf, M. J., & de Bont, J. A. (1990). Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase. Applied and Environmental Microbiology, 56, 1347–1351.

    CAS  Google Scholar 

  5. Archelas, A., & Furstoss, R. (1997). Synthesis of enantiopure epoxides through biocatalytic approaches. Annual Review of Microbiology, 51, 491–525.

    Article  CAS  Google Scholar 

  6. Choi, W. J. (2009). Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution. Applied microbiology and biotechnology., 84, 239–247.

    Article  CAS  Google Scholar 

  7. Lin, H., Liu, J. Y., Wang, H. B., Ahmed, A. A., & Wu, Z. L. (2011). Biocatalysis as an alternative for the production of chiral epoxides: a comparative review. Journal of Molecular Catalysis B: Enzymatic., 72, 77–89.

    Article  CAS  Google Scholar 

  8. Fukami, T., Katoh, M., Yamazaki, H., Yokoi, T., & Nakajima, M. (2008). Human cytochrome P450 2A13 efficiently metabolizes chemicals in air pollutants: naphthalene, styrene, and toluene. Chemical Research in Toxicology, 21, 720–725.

    Article  CAS  Google Scholar 

  9. Green, J., & Dalton, H. (1989). A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath). The Biochemical Journal, 259, 167–172.

    Article  CAS  Google Scholar 

  10. Thibodeaux, C. J., Chang, W. C., & Liu, H. W. (2012). Enzymatic chemistry of cyclopropane, epoxide, and aziridine biosynthesis. Chemical Reviews, 112, 1681–1709.

    Article  CAS  Google Scholar 

  11. van Berkel, W. J., Kamerbeek, N. M., & Fraaije, M. W. (2006). Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. Journal of Biotechnology, 124, 670–689.

    Article  Google Scholar 

  12. Huijbers, M. M., Montersino, S., Westphal, A. H., Tischler, D., & van Berkel, W. J. (2014). Flavin dependent monooxygenases. Archives of Biochemistry and Biophysics., 544C, 2–17.

    Article  Google Scholar 

  13. Panke, S., Witholt, B., Schmid, A., & Wubbolts, M. G. (1998). Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Applied and Environmental Microbiology, 64, 2032–2043.

    CAS  Google Scholar 

  14. Teufel, R., Mascaraque, V., Ismail, W., Voss, M., Perera, J., Eisenreich, W., Haehnel, W., & Fuchs, G. (2010). Bacterial phenylalanine and phenylacetate catabolic pathway revealed. Proceedings of the National Academy of Sciences of the United States of America, 107, 14390–14395.

    Article  CAS  Google Scholar 

  15. Montersino, S., Tischler, D., Gassner, G. T., & van Berkel, W. J. (2011). Catalytic and structural features of flavoprotein hydroxylases and epoxidases. Advanced Synthesis and Catalysis, 353, 2301–2319.

    Article  CAS  Google Scholar 

  16. Morrison, E., Kantz, A., Gassner, G. T., & Sazinsky, M. H. (2013). Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction. Biochemistry, 52, 6063–6075.

    Article  CAS  Google Scholar 

  17. Ukaegbu, U. E., Kantz, A., Beaton, M., Gassner, G. T., & Rosenzweig, A. C. (2010). Structure and ligand binding properties of the epoxidase component of styrene monooxygenase. Biochemistry, 49, 1678–1688.

    Article  CAS  Google Scholar 

  18. Kantz, A., & Gassner, G. T. (2011). Nature of the reaction intermediates in the flavin adenine dinucleotide-dependent epoxidation mechanism of styrene monooxygenase. Biochemistry, 50, 523–532.

    Article  CAS  Google Scholar 

  19. Kantz, A., Chin, F., Nallamothu, N., Nguyen, T., & Gassner, G. T. (2005). Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase. Archives of Biochemistry and Biophysics., 442, 102–116.

    Article  CAS  Google Scholar 

  20. Bae, J. W., Doo, E. H., Shin, S. H., Lee, S. G., Jeong, Y. J., Park, J. B., & Park, S. (2010). Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source. Process Biochemistry., 45, 147–152.

    Article  CAS  Google Scholar 

  21. Tischler, D., Eulberg, D., Lakner, S., Kaschabek, S. R., van Berkel, W. J., & Schlomann, M. (2009). Identification of a novel self-sufficient styrene monooxygenase from Rhodococcus opacus 1CP. Journal of Bacteriology, 191, 4996–5009.

    Article  CAS  Google Scholar 

  22. Tischler, D., Kermer, R., Groning, J. A., Kaschabek, S. R., van Berkel, W. J., & Schlomann, M. (2010). StyA1 and StyA2B from Rhodococcus opacus 1CP: a multifunctional styrene monooxygenase system. Journal of Bacteriology, 192, 5220–5227.

    Article  CAS  Google Scholar 

  23. Tischler, D., Schlomann, M., van Berkel, W. J., & Gassner, G. T. (2013). FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase. FEBS letters., 587, 3848–3852.

    Article  CAS  Google Scholar 

  24. Tischler, D., Groning, J. A., Kaschabek, S. R., & Schlomann, M. (2012). One-component styrene monooxygenases: an evolutionary view on a rare class of flavoproteins. Applied Biochemistry and Biotechnology, 167, 931–944.

    Article  CAS  Google Scholar 

  25. Munro, A. W., Leys, D. G., McLean, K. J., Marshall, K. R., Ost, T. W., Daff, S., Miles, C. S., Chapman, S. K., Lysek, D. A., Moser, C. C., Page, C. C., & Dutton, P. L. (2002). P450 BM3: the very model of a modern flavocytochrome. Trends in Biochemical Sciences, 27, 250–257.

    Article  CAS  Google Scholar 

  26. Jawanda, N., Ahmed, K., & Tu, S. C. (2008). Vibrio harveyi flavin reductase–luciferase fusion protein mimics a single-component bifunctional monooxygenase. Biochemistry, 47, 368–377.

    Article  CAS  Google Scholar 

  27. Sambrook, J., & Russell, D. W. (2001). Molecular cloning; a laboratory manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  28. Oelschlagel, M., Groning, J. A., Tischler, D., Kaschabek, S. R., & Schlomann, M. (2012). Styrene oxide isomerase of Rhodococcus opacus 1CP, a highly stable and considerably active enzyme. Applied and Environmental Microbiology, 78, 4330–4337.

    Article  Google Scholar 

  29. O’Connor, K. E., & Hartmans, S. (1998). Indigo formation by aromatic hydrocarbon-degrading bacteria. Biotechnology Letters., 20, 219–233.

    Article  Google Scholar 

  30. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D. et al. (2005). Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Ed.), The Proteomics Protocols Handbook (pp. 571–607). Humana Press.

  31. Chen, R. F. (1967). Removal of fatty acids from serum albumin by charcoal treatment. The Journal of Biological Chemistry, 242, 173–181.

    CAS  Google Scholar 

  32. Motulsky, H. J., & Christopoulos, A. (2003). Fitting models to biological data using linear and nonlinear regression. San Diego: GraphPad Software Inc..

    Google Scholar 

  33. Choi, H. S., Kim, J. K., Cho, E. H., Kim, Y. C., Kim, J. I., & Kim, S. W. (2003). A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli. Biochemical and biophysical research communications., 306, 930–936.

    Article  CAS  Google Scholar 

  34. Otto, K., Hofstetter, K., Rothlisberger, M., Witholt, B., & Schmid, A. (2004). Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase. Journal of Bacteriology, 186, 5292–5302.

    Article  CAS  Google Scholar 

  35. Qaed, A. A., Lin, H., Tang, D. F., & Wu, Z. L. (2011). Rational design of styrene monooxygenase mutants with altered substrate preference. Biotechnology Letters, 33, 611–616.

    Article  CAS  Google Scholar 

  36. Lin, H., Qiao, J., Liu, Y., & Wu, Z. L. (2010). Styrene monooxygenase from Pseudomonas sp. LQ26 catalyzes the asymmetric epoxidation of both conjugated and unconjugated alkenes. Journal of Molecular Catalysis B: Enzymatic., 67, 236–241.

    Article  CAS  Google Scholar 

  37. Lin, H., Liu, Y., & Wu, Z. L. (2011). Highly diastereo- and enantio-selective epoxidation of secondary allylic alcohols catalyzed by styrene monooxygenase. Chemical Communications (Cambridge, England), 47, 2610–2612.

    Article  CAS  Google Scholar 

  38. Lin, H., Tang, D. F., Ahmed, A. A., Liu, Y., & Wu, Z. L. (2012). Mutations at the putative active cavity of styrene monooxygenase: enhanced activity and reversed enantioselectivity. Journal of Biotechnology, 161, 235–241.

    Article  CAS  Google Scholar 

  39. De Mot, R., & Parret, A. H. (2002). A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes. Trends in Microbiology, 10, 502–508.

    Article  CAS  Google Scholar 

  40. Kapadia, G. J., Tokuda, H., Sridhar, R., Balasubramanian, V., Takayasu, J., Bu, P., Enjo, F., Takasaki, M., Konoshima, T., & Nishino, H. (1998). Cancer chemopreventive activity of synthetic colorants used in foods, pharmaceuticals and cosmetic preparations. Cancer Letters, 129, 87–95.

    Article  CAS  Google Scholar 

  41. Hössel, R. (1999a). Synthese von Derivaten des Indirubins und Untersuchungen zur Mechanismsaufklärung ihrer antineoplastischen Wirkung. Kaiserslautern: Universität Kaiserslautern.

    Google Scholar 

  42. Guengerich, F. P., Sorrells, J. L., Schmitt, S., Krauser, J. A., Aryal, P., & Meijer, L. (2004). Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis. Journal of Medicinal Chemistry, 47, 3236–3241.

    Article  CAS  Google Scholar 

  43. Guengerich, P. F., Martin, M. V., McCormick, W. A., Nguyenb, L. P., Glover, E., & Bradfield, C. A. (2004). Aryl hydrocarbon receptor response to indigoids in vitro and in vivo. Archives of Biochemistry and Biophysics., 423, 309–316.

    Article  CAS  Google Scholar 

  44. Wu, Z. L., Aryal, P., Lozach, O., Meijer, L., & Guengerich, F. P. (2005). Biosynthesis of new indigoid inhibitors of protein kinases using recombinant cytochrome P450 2A6. Chemistry & Biodiversity, 2, 51–65.

    Article  CAS  Google Scholar 

  45. Harrer, R. (2012). Indigo auf Speicherchips. Chemie in userer Zeit, 46, 136.

    Article  Google Scholar 

  46. Uehara, K., Takagishi, K., & Tanaka, M. (1987). The Al/Indigo/Au photovoltaic cell. Solar Cells., 22, 295–301.

    Article  CAS  Google Scholar 

  47. Lüttke, W., & Hunsdiecker, D. (1966). Theoretische und spetroskopische Untersuchungen an Indigofarbstoffen, IV. Substituenteneffekt am Indigo: Die Darstellung des 5.5′-und 6.6′-Diaza-indigos. Chemische Berichte, 99, 2146–2154.

    Article  Google Scholar 

  48. Gursky, L. J., Nikodinovic-Runic, J., Feenstra, K. A., & O’Connor, K. E. (2010). In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis. Applied microbiology and biotechnology., 85, 995–1004.

    Article  CAS  Google Scholar 

  49. Nikodinovic-Runic, J., Coulombel, L., Francuski, D., Sharma, N. D., Boyd, D. R., Ferrall, R. M., & O’Connor, K. E. (2013). The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3. Applied microbiology and biotechnology., 97, 4849–4858.

    Article  CAS  Google Scholar 

  50. Baggi, G., Boga, M. M., Catelani, D., Galli, E., & Treccani, V. (1983). Styrene catabolism by a strain of Pseudomonas fluorescens. Systematic and Applied Microbiology, 4, 141–147.

    Article  CAS  Google Scholar 

  51. Wehmeier Personal Communication.

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Acknowledgments

This work was supported by NIHSC1 GM081140 to George Gassner and Dirk Tischler was supported by a Fulbright Scholarship. Nonye Okonkwo and Berhanegebriel Assefa were supported by the NIH-MARC program at SFSU.

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Authors and Affiliations

  1. TU Bergakademie Freiberg, Freiberg, Germany

    Thomas Heine, Catleen Conrad, Anika Scholtissek, Michael Schlömann & Dirk Tischler

  2. San Francisco State University, San Francisco, CA, USA

    Kathryn Tucker, Nonye Okonkwo, Berhanegebriel Assefa, George Gassner & Dirk Tischler

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  1. Thomas Heine
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  2. Kathryn Tucker
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  3. Nonye Okonkwo
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Correspondence to George Gassner or Dirk Tischler.

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Heine, T., Tucker, K., Okonkwo, N. et al. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins. Appl Biochem Biotechnol 181, 1590–1610 (2017). https://doi.org/10.1007/s12010-016-2304-4

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