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Challenges in capturing oxygenase activity in vitro

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Journal of the American Oil Chemists' Society

Abstract

Biocatalysis using oxygenase or desaturase enzymes has the potential to add value to native fats and oils by adding oxygen, hydroxyl groups, or double bonds to create regio- and/or stereospecific products. These enzymes are a subset of the large class of oxidoreductase enzymes (from EC subgroups 1.13 and 1.14) involved with biological oxidation and reduction. In vitro biocatalytic processing using these enzymes is hampered by the high cost of the stoichiometric cofactors. This article reviews recent progress in developing in vitro redox enzyme biocatalysis for commercial-scale syntheses. Coenzyme recycling and electrochemical redox cycling as methods for cofactor regeneration are described and commercial applications indicated. Direct charge transfer without use of mediators is described as the cleanest way of introducing the reducing power into the catalytic cycle. Our electrochemically driven cytochrome P450cam bioreactor is discussed as an example of direct charge transfer to a redox protein. Site-directed mutagenesis in the active site of the P450cam monooxygenase greatly improved performance for the conversion of the nonnative substrate, styrene to styrene oxide. This epoxidation reaction was also shown to give a single product (styrene oxide) in the bioelectrochemical reactor when the diatomic oxygen co-substrate was managed properly.

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References

  1. Shanklin, J., and E.B. Cahoon, Desaturation and Related Modifications of Fatty Acids, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611–641 (1998).

    Article  CAS  Google Scholar 

  2. Lipscomb, J.D., Biochemistry of the Soluble Methane Monooxygenase, Annu. Rev. Microbiol. 48:371–399 (1994).

    Article  CAS  Google Scholar 

  3. Lee, L.G., and G.M. Whitesides, Enzyme-Catalyzed Organic Synthesis: A Comparison of Strategies for in Situ Regeneration of NAD from NADH, J. Am. Chem. Soc. 107:6999–7008 (1985).

    Article  CAS  Google Scholar 

  4. Whitesides, G.M., and C.H. Wong, Enzymes as Catalysts in Organic Synthesis, Ang. Chem. Int. Ed. 24:617–718 (1985).

    Article  Google Scholar 

  5. Rover, L., Jr., J.C.B. Fernandes, G. de Oleverio Neto, L.T. Kubota, E. Katekawa, and S.H.P. Serrano, Study of NADH Stability Using Ultraviolet-Visible Spectrophotometric Analysis and Factorial Design, Anal. Biochem. 260:50–55 (1998).

    Article  CAS  Google Scholar 

  6. Chenault, H.K., and G.M. Whitesides, Regeneration of Nicotinamide Cofactors for Use in Organic Synthesis, Applied Biochem. Biotechnol. 14:147–197 (1987).

    CAS  Google Scholar 

  7. Devaux-Basseguy, R., A. Bergel, and M. Comtat, Potential Applications of NAD(P)-Dependent Oxidoreductases in Synthesis: A Survey, Enzyme Microb. Technol. 20:248–258 (1997).

    Article  CAS  Google Scholar 

  8. Brielbeck, B., M. Frede, and E. Steckhan, Continuous Electroenzymatic Synthesis Employing the Electrochemical Enzyme Membrane Reactor, Biocatalysis 10:49–64 (1994).

    CAS  Google Scholar 

  9. Bommarius, A.S., M. Schwarm, and K. Drauz, Biocatalysis to Amino Acid-Based Chiral Pharmaceuticals—Examples and Perspectives, J. Molec. Catalysis B. Enzymatic 5:1–11 (1997).

    Article  Google Scholar 

  10. Cantet, J., A. Bergel, and M. Comtat, Coupling of the Electroenzymatic Reduction of NAD+ with a Synthesis Reaction, Enzyme Microb. Technol. 18:72–79 (1996).

    Article  CAS  Google Scholar 

  11. Nidetzky, B., W. Neuhauser, and D. Haltrich, Continuous Enzymatic Production of Xylitol with Simultaneous Coenzyme Regeneration in a Charged Membrane Reactor, Biotechnol. Bioengr. 52:387–396 (1996).

    Article  CAS  Google Scholar 

  12. Jaegfeldt, H., Study of the Products Formed in the Electrochemical Reduction of Nicotinamide Adenine Dinucleotide, Bioelectrochem. Bioenerg. 8:355–370 (1981).

    Article  CAS  Google Scholar 

  13. Long, T.Y., and Y.H. Chen, Electrochemical Regeneration of Coenzyme NADH on a Histidine Modified Silver Electrode, J. Electroanalyt. Chem. 440:239–242 (1997).

    Article  CAS  Google Scholar 

  14. Yun, S.E., M. Taya, and S. Tone, Direct Reduction of NAD+ by Electrochemical Procedure and Application of the Regenerated NADH to Enzyme Reaction, Biotechnol. Lett. 16:1053–1058 (1994).

    Article  CAS  Google Scholar 

  15. Steckhan, E., Electroenzymatic Synthesis, in Topics in Current Chemistry: Electrochemistry V, edited by E. Steckhan, Springer-Verlag, Berlin, 1994, pp. 83–111.

    Google Scholar 

  16. Ruppert, R., S. Herrmann, and E. Steckhan, Efficient Indirect Electrochemical in-Situ Regeneration of NADH: Electrochemically Driven Enzymatic Reduction of Pyruvate Catalyzed D-LDH, Tetrahedron Lett. 28:6583–6586 (1987).

    Article  CAS  Google Scholar 

  17. Cosnier, S., and H. Gunther, A Polypyrrole [RhIII(C5Me5)(bpy)Cl]+ Modified Electrode for the Reduction of NAD+ Cofactor: Application to the Enzymatic Reduction of Pyruvate, J. Electroanalyt. Chem. 315:307–312 (1991).

    Article  CAS  Google Scholar 

  18. Franke, M., and E. Steckhan, Tris(2,2′-bipyridyl-5-sulfonic acid)rhodium(iii), An Improved Redox Catalyst for the Light-Induced and the Electrochemically Initiated Enzymatic Reduction of Carbonyl-Compounds, Angew. Chem. Int. Ed. Engl. 27:265–267 (1988).

    Article  Google Scholar 

  19. Langen, R., I.J. Chang, J.P. Germanas, J.H. Richerds, J.R. Winkler, and H.B. Gray, Electron Tunneling in Proteins: Coupling Through a Beta Strand, Science 268:1733–1735 (1995).

    Article  CAS  Google Scholar 

  20. Heller, A., Electrical Wiring of Redox Enzymes, Acc. Chem. Res. 23:128–134 (1990).

    Article  CAS  Google Scholar 

  21. Wong, L.S., V.L. Vilker, W.T. Yap, and V. Reipa, Characterization of the Mercaptoethyl-Amine-Modified Gold Electrode Surface and Analyses of the Direct Electron Transfer to Putidaredoxin, Langmuir 11:4818–4822 (1995).

    Article  CAS  Google Scholar 

  22. Armstrong, F.A., Probing Metalloproteins by Voltammetry, Struct. Bonding (Berlin) 72:139–221 (1990).

    Google Scholar 

  23. Bond, A.M., Chemical and Electrochemical Approaches to the Investigation of Redox Reactions of Simple Electron Transfer Metalloproteins, Inorg. Chem. Acta 226:293–340 (1994).

    Article  CAS  Google Scholar 

  24. Ruzgas, T., L. Wong, A.K. Gaigalas, and V.L. Vilker, Electron Transfer Between Surface-Confined Cytochrome c and N-Acetyl Cysteine Modified Gold Electrode, Langmuir 14:7298–7305 (1998).

    Article  CAS  Google Scholar 

  25. Taniguchi, I., Probing Metalloproteins and Bioelectrochemical Systems, Interface 6:34–37 (1997).

    CAS  Google Scholar 

  26. Hawkridge, F.M., and I. Taniguchi, The Direct Electron-Transfer Reactions of Cytochrome c at Electrode Surfaces, Comm. Inorg. Chem. 17:163–187 (1995).

    CAS  Google Scholar 

  27. Allen, P.M., H.A.O. Hill, and N.J. Walton, Surface Modifiers for the Promotion of Direct Electrochemistry of Cytochrome c, J. Electroanalyt. Chem. 178:69–86 (1984).

    Article  CAS  Google Scholar 

  28. Szucs, A., and M. Novak, Stable and Reversible Electrochemistry of Cytochrome c on Bare Electrodes: Part I Effect of Ionic Strength, Ibid.:75–84 (1995).

    Article  Google Scholar 

  29. Reipa, V., A.K. Gaigalas, and V.L. Vilker, Spectroscopic Realtime Ellipsometry of Putidaredoxin Adsorption on Gold Electrodes, Langmuir 13:3508–3514 (1997).

    Article  CAS  Google Scholar 

  30. Reipa, V., A.K. Gaigalas, J. Edwards, and V.L. Vilker, Surface Enhanced Raman Spectroscopy (SERS) Evidence of Charge Transfer Between Putidaredoxin and a Silver Electrode, J. Electroanalyt. Chem. 395:299–303 (1995).

    Article  Google Scholar 

  31. Gaigalas, A.K., V. Reipa, and V.L. Vilker, Observation of Electron Transfer Between a Silver Electrode and Putidaredoxin Using Electromodulated Reflectance Spectroscopy, J. Colloid Interface Sci. 186:339–351 (1997).

    Article  CAS  Google Scholar 

  32. Niwa, K., M. Furukawa, and K. Niki, IR Reflectance Studies of Electron Transfer Promoters for Cytochrome c on a Gold Electrode, J. Electroanalyt. Chem. 245:275–285 (1988).

    Article  CAS  Google Scholar 

  33. Kazlauskaite, J., A.C.G. Westlake, L.-L. Wong, and H.A.O. Hill, Direct Electrochemistry of Cytochrome P450cam, Chem. Commun.:2189–2190 (1996).

  34. Mueller, E.J., P.J. Loida, and S.G. Sligar, Twenty-five Years of P450cam Research, in Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edn., edited by P.R. Ortiz de Montellano, Plenum Press, New York, 1995, pp. 83–124.

    Google Scholar 

  35. Grayson, D.A., and V.L. Vilker, Kinetic Characterization of Chiral Biocatalysis of Cycloarenes by the Camphor 5-Monooxygenase Enzyme System. J. Molecular Catalysis: Enzymatic 6:533–547 (1999).

    Article  CAS  Google Scholar 

  36. Schneider, S., M.G. Wubbolts, D. Sanglard, and B. Wiltholt, Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for in Vivo Application of Cytochrome P-450BM-3 Monooxygenase, Appl. Environ. Microbiol. 64: 3784–3790 (1998).

    CAS  Google Scholar 

  37. Castro, C.E., W.H. Yokoyama, and N.O. Belser, Biodehalogenation, Reductive Reactivities of Microbial and Mammalian Cytochromes P-450 Compared with Heme and Whole Cell Models, J. Agric. Food Chem. 36:915–919 (1988).

    Article  CAS  Google Scholar 

  38. Estabrook, R.W., K.M. Faulkner, M.S. Shet, and C.W. Fisher, Application of Electrochemistry for P450-Catalyzed Reactions, Methods Enzymol. 272:44–50 (1996).

    CAS  Google Scholar 

  39. Ravichandran, K.G., S.S. Boddupalli, C.A. Hasemann, J.A. Peterson, and J. Deisenhofer, Crystal Structure of Hemoprotein Domain of P450BM-3, a Prototype for Microsomal P450s, Science 261:731–736 (1993).

    Article  CAS  Google Scholar 

  40. Roitberg, A.E., M.J. Holden, M.P. Mayhew, I.V. Kurnikov, D.N. Beratan, and V.L. Vilker, Binding and Electron Transfer Between Putidaredoxin and Cytochrome P450cam (CYP101), Theory and Experiments, J. Am. Chem. Soc. 120:8927–8932 (1998).

    Article  CAS  Google Scholar 

  41. Reipa, V., M.P. Mayhew, and V.L. Vilker, A Direct-Electrode-Driven P-450 Cycle for Biocatalysis, Proc. Natl. Acad. Sci. USA 94:13554–13558 (1997).

    Article  CAS  Google Scholar 

  42. Fruetel, J.A., J.R. Collins, D.L. Camper, G.H. Loew, and P.R. Ortiz de Montellano, Calculated and Experimental Absolute Stereochemistry of the Styrene and β-Methylstyrene Epoxides Formed by Cytochrome P450cam, J. Am. Chem. Soc. 114:6987–6993 (1992).

    Article  CAS  Google Scholar 

  43. Lvov, Y.M., Z. Lu, J.B. Schenkman, X. Zu, and J.F. Rusling, Direct Electrochemistry of Myoglobin and Cytochrome P450cam in Alternate Layer-by-Layer Films with DNA and Other Polyions, Ibid.:4073–4080 (1997).

    Article  Google Scholar 

  44. Nickerson, D.P., C.F. Harford-Cross, S. Fulcher, and L.-L. Wong, The Catalytic Activity of Cytochrome P450cam Towards Styrene Oxidation Is Increased by Site-Directed Mutagenesis, FEBS Letters 405:153–156 (1997).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Biotechnology Division, National Institute of Standards and Technology, 20899-8312, Gaithersburg, Maryland

    Vincent L. Vilker, Vytas Reipa, Martin Mayhew & Marcia J. Holden

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  1. Vincent L. Vilker
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  2. Vytas Reipa
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  3. Martin Mayhew
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  4. Marcia J. Holden
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Correspondence to Vincent L. Vilker.

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Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the material or equipment is necessarily the best available for the purpose.

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Vilker, V.L., Reipa, V., Mayhew, M. et al. Challenges in capturing oxygenase activity in vitro . J Amer Oil Chem Soc 76, 1283–1289 (1999). https://doi.org/10.1007/s11746-999-0140-1

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  • DOI: https://doi.org/10.1007/s11746-999-0140-1

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