Abstract
Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family1 of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis2,3,4,5, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds6. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals7,8,9,10. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution11 of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the ‘peroxide shunt’ pathway12,13 with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Nelson, D. R. et al. . P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42 (1996).
Cerniglia, C. E. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351–368 (1992).
Cook, D. L. & Atkins, W. M. Enhanced detoxication due to distributive catalysis and toxic thresholds: a kinetic analysis. Biochemistry 36, 10801–10806 (1997).
Waxman, D. J. & Chang, T. K. H. in Cytochrome P450: Structure, Mechanism and Biochemistry 2nd edn(ed. Ortiz de Montellano, P. R.) 391–417 (Plenum, New York, 1995).
Gonzalez, F. J. & Nebert, D. W. Evolution of the P450-gene superfamily—animal plant warfare, molecular drive and human genetic differences in drug oxidation. Trends Genet. 6, 182–186 (1990).
Sono, M., Roach, M. P., Coulter, E. D. & Dawson, J. H. Heme-containing oxygenases. Chem. Rev. 96, 2841–2887 (1996).
Jounaidi, Y., Hecht, J. E. D. & Waxman, D. J. Retroviral transfer of human cytochrome P450 genes for oxazaphosphorine-based cancer gene therapy. Cancer Res. 58, 4391–4401 (1998).
Wackett, L. P., Sadowsky, M. J., Newman, L. M., Hur, H.-G. & Li, S. Metabolism of polyhalogenated compounds by a genetically engineered bacterium. Nature 368, 627–629 (1994).
Faber, K. Biotransformations in Organic Chemistry 208–220 (Springer, Berlin, 1997).
Duport, C., Spagnoli, R., Degryse, E. & Pompon, D. Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast. Nature Biotechnol. 16, 186–189 (1998).
Arnold, F. H. Design by directed evolution. Acc. Chem. Res. 31, 125–131 (1998).
Nordblom, G. D., White, R. E. & Coon, M. J. Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P450. Arch. Biochem. Biophys. 175, 524–533 (1976).
Hrycay, E. G., Gustafsson, J.-A., Ingelman-Sundberg, M. & Ernster, L. Sodium periodate, sodium chlorite, organic hydroperoxides and hydrogen peroxide as hydroxylating agents in steroid hydroxylation reactions catalyzed by partially purified cytochrome P450. Biochem. Biophys. Res. Commun. 66, 209–216 (1975).
Hummel, W. & Kula, M.-R. Dehydrogenases for the synthesis of chiral compounds. Eur. J. Biochem. 184, 1–13 (1989).
Seelbach, K. et al. . Anovel, efficient regenerating method of NADPH using a new formate dehydrogenase. Tetrahedron Lett. 37, 1377–1380 (1997).
Henriksen, A. et al. . Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography. Biochemistry 37, 8054–8060 (1998).
Lin, Z., Thorsen, T. & Arnold, F. H. Functional expression of horseradish peroxidase in Escherichia coli by directed evolution. Biotechnol. Prog. 15, 467–471 (1999).
Gunsalus, I. C., Meeks, J. R., Lipscomb, J. D., Debrunner, P. G. & Munck, E. in Molecular Mechanisms of Oxygen Activation(ed. Hayaishi, O.) 559–613 (Academic, New York, 1974).
England, P. A., Harford-Cross, C. F., Stevenson, J.-A., Rouch, D. A. & Wong, L.-L. The oxidation of naphthalene and pyrene by cytochrome P450cam. FEBS Lett. 424, 271–274 (1998).
Poulos, T. L. & Raag, R. Cytochrome P450cam: crystallography, oxygen activation, and electron transfer. FASEB J. 6, 674–679 (1992).
Graham-Lorence, S. & Peterson, J. A. P450s: Structural similarities and functional differences. FASEB J. 10, 206–214 (1996).
Denu, J. M. & Tanner, K. G. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 37, 5633–5642 (1998).
van Deurzen, M. P. J., van Rantwijk, F. & Sheldon, R. A. Selective oxidations catalyzed by peroxidases. Tetrahedron 53, 13183–13220 (1997).
Crameri, A., Raillard, S.-A., Bermudez, E. & Stemmer, W. P. C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).
Zhao, H., Giver, L., Shao, Z., Affholter, J. A. & Arnold, F. H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnol. 16, 258–261 (1998).
Moore, J. C. & Arnold, F. H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nature Biotechnol. 14, 458–467 (1996).
Giver, L., Gershenson, A., Freskgard, P. O. & Arnold, F. H. Directed evolution of a thermostable esterase. Proc. Natl Acad. Sci. USA 95, 12809–12813 (1998).
Zhao, H. & Arnold, F. H. Directed evolution converts subtillisin E into a functional equivalent of thermitase. Protein Eng. 12, 47–53 (1999).
McKay, C. P. & Hartman, H. Hydrogen peroxide and the evolution of oxygenic photosynthesis. Origins Life Evol. Biosphere 21, 157–163 (1991).
Samuilov, V. D. Photosynthetic oxygen: the role of H2O2. A review. Biochemistry (Moscow) 62, 451–454 (1997).
Zhao, H., Moore, J. C., Volkov, A. A. & Arnold, F. H. in Manual of Industrial Microbiology and Biotechnology 2nd edn(eds Demain, A. L. & Davies, J. E.) 597–604 (ASM Press, Washington DC, 1999).
Unger, B. P., Gunsalus, I. C. & Sligar, S. G. Nucleotide-sequence of the Pseudomonas-putida cytochrome P-450camgene and its expression in Escherichia-coli . J. Biol. Chem. 261, 1158–1163 (1986).
Acknowledgements
We thank J. H. Richards for encouragement and support, P. R. Ortiz de Montellano for discussions, and G. Bandara for assistance with purification. This work was supported by the Biotechnology Research and Development Corporation and by the Office of Naval Research. H.J. received partial support from the Korea Research Foundation.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Joo, H., Lin, Z. & Arnold, F. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670–673 (1999). https://doi.org/10.1038/21395
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/21395
This article is cited by
-
Current state and future perspectives of engineered and artificial peroxygenases for the oxyfunctionalization of organic molecules
Nature Catalysis (2020)
-
Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119
JBIC Journal of Biological Inorganic Chemistry (2020)
-
Structural Insights into Catalytic Versatility of the Flavin-dependent Hydroxylase (HpaB) from Escherichia coli
Scientific Reports (2019)
-
Engineered global regulator H-NS improves the acid tolerance of E. coli
Microbial Cell Factories (2018)
-
From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution
Applied Microbiology and Biotechnology (2018)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.