Cloning, Expression and Identification of the Enzyme P450-SCA12 from Streptomyces cavourensis YBQ59
Main Article Content
Abstract
The use of microbial cytochrome P450 as a biocatalyst offers significant potential for the development of natural compounds due to its ability to transform a wide range of structures with high regio- and stereoselectivity. In this study, a 1263 bp gene from S. cavourensis strain YBQ59, encoding the CYP154C subfamily enzyme P450-SCA12, was successfully expressed in E. coli CD43(DE3). The enzyme P450-SCA12 was produced in a soluble form when fused with thioredoxin (Trx) and 6xHis proteins from the pET32b(+) vector. After purification through ion affinity chromatography using a His-select® cobalt affinity gel column, the enzyme P450-SCA12 was obtained in an active form, with a molecular weight of approximately 63 kDa and a yield of around 1000 nmol/L from the Terrific Broth culture medium. Screening of substrates revealed that this enzyme specifically metabolizes three steroid compounds: testosterone, nandrolone, and 4-androstenedione. These findings highlight the potential of the P450-SCA12 enzyme from the actinomycete S. cavourensis YBQ59 in natural product development, particularly for metabolizing steroid compounds to create new steroid-based drugs.
Keywords:
P450, steroids, CYP154C3, Streptomyces cavourensis, YBQ59.
References
[1] G. D. Nardo, G. Gilardi, Natural Compounds as Pharmaceuticals: The Key Role of Cytochromes P450 Reactivity, Trends Biochem. Sci., Vol. 45, 2020, pp. 511-525, https://doi.org/10.1016/j.tibs.2020.03.004.
[2] J. A. McIntosh, C. C. Farwell, F. H. Arnold, Expanding P450 Catalytic Reaction Space through Evolution and Engineering, Curr. Opin. Chem. Biol., Vol. 19, 2014, pp. 126-134, https://doi.org/10.1016/j.cbpa.2014.02.001.
[3] D. R. Nelson, Cytochrome P450 Diversity in the Tree of Life, Biochim, Biophys, Acta Proteins Proteomics, Vol. 1866, 2018, pp. 141-154, https://doi.org/10.1016/j.bbapap.2017.05.003.
[4] M. Moir, J. J. Danon, T. A. Reekie, M. Kassiou, An Overview of Late-Stage Functionalization in Today’s Drug Discovery, Expert Opin, Drug Discov., Vol. 14, 2019, pp. 1137-1149, https://doi.org/10.1080/17460441.2019.1653850.
[5] M. A. Cho, S. Han, Y. R. Lim, V. Kim, H. Kim, D. Kim, Streptomyces Cytochrome P450 Enzymes and Their Roles in the Biosynthesis of Macrolide Therapeutic Agents, Biomol. Ther., Vol. 27, 2019, pp. 127-133,
https://doi.org/10.4062%2Fbiomolther.2018.183.
[6] D. C. Lamb, F. P. Guengerich, S. L. Kelly, M. R. Waterman, Exploiting Streptomyces Coelicolor A3(2) P450s as a Model for Application in Drug Discovery, Expert Opin. Drug Metab. Toxicol., Vol. 2, 2006, pp. 27-40,
https://doi.org/10.1517/17425255.2.1.27.
[7] J. D. Rudolf, C. Y. Chang, M. Ma, B. Shen, Cytochromes P450 for Natural Product Biosynthesis in Streptomyces: Sequence, Structure, and Function, Nat. Prod. Rep., Vol. 34, 2017, pp. 1141-1172,
https://doi.org/10.1039/c7np00034k.
[8] A. Worsch, F. K. Eggimann, M. Girhard, C. J. von Bühler, F. Tieves, R. Czaja, A. Vogel, C. Grumaz, K. Sohn, S. Lütz, M. Kittelmann, V. B. Urlacher, A Novel Cytochrome P450 Mono-Oxygenase from Streptomyces Platensis Resembles Activities of Human Drug Metabolizing P450s, Biotechnol. Bioeng., Vol. 115, 2018, pp. 2156-2166,
https://doi.org/10.1002/bit.26781.
[9] N. Kanoh, A. K. Asano, K. Suzuki, Y. Takahashi, T. Miyazawa, T. Nakamura, T. Moriya,
H. Hirano, H. Osada, Y. Iwabuchi, S. Takahashi, An Integrated Screening System for the Selection of Exemplary Substrates for Natural and Engineered Cytochrome P450s, Sci. Rep., Vol. 9, 2019, pp. 18023, https://doi.org/10.1038/s41598-019-54473-8.
[10] H. N. T. Vu, D. T. Nguyen, H. Q. Nguyen, H. H. Chu, S. K. Chu, M. Van Chau, Q. T. Phi, Antimicrobial and Cytotoxic Properties of Bioactive Metabolites Produced by Streptomyces cavourensis YBQ59 Isolated from Cinnamomum cassia Prels in Yen Bai Province of Vietnam, Curr. Microbiol., Vol. 75, 2018, pp. 1247-1255, https://doi.org/10.1007/s00284-018-1517-x.
[11] T. Omura, R. Sato, The Carbon Monoxide-Binding Pigment of Liver Microsomes, J. Biol. Chem., Vol. 239, 1964, pp. 2370-2378, https://doi.org/10.1016/S0021-9258(20)82244-3.
[12] K. C. Leibman, A. G. Hildebrandt, R. M. Estabrook, Spectrophotometric Studies of Interactions Between Various Substrates in Their Binding to Microsomal Cytochrome P-450, Biochem. Biophys. Res. Commun., Vol. 36, 1969, pp. 789-794, https://doi.org/10.1016/0006-291X(69)90678-0.
[13] T. T. B. Ly, Y. Khatri, J. Zapp, M. C. Hutter, R. Bernhardt, CYP264B1 from Sorangium cellulosum So ce56: A Fascinating Norisoprenoid and Sesquiterpene Hydroxylase, Appl. Microbiol. Biotechnol., Vol. 95, 2012, pp. 123-133, https://doi.org/10.1007/s00253-011-3727-z.
[14] P. Subedi, K. H. Kim, Y. S. Hong, J. H. Lee, T. J. Oh, Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 from Streptomyces Species, J. Microbiol. Biotechnol., Vol. 31, 2021, pp. 464-474, https://doi.org/10.4014%2Fjmb.2010.10020.
[15] J. McCoy, E. LaVallie, Expression and Purification of Thioredoxin Fusion Proteins, Curr. Protoc. Mol. Biol., Vol. 28, 1994, pp. 16-8, https://doi.org/10.1002/0471140864.ps0607s10.
[16] T. Makino, Y. Katsuyama, T. Otomatsu, N. Misawa, Y. Ohnishi, Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces Griseus Cytochrome P450 CYP154C3, Appl, Environ. Microbiol., Vol. 80, 2013, pp. 1371-1379, https://doi.org/10.1128/aem.03504-13.
[17] M. A. Kaderbhai, C. C. Ugochukwu, S. L. Kelly, D. C. Lamb, Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia Coli, J. Appl. Environ. Microbiol., Vol. 67, 2001, pp. 2136-2138, https://doi.org/10.1128/aem.67.5.2136-2138.200.
[18] M. Taylor, D. C. Lamb, R. Cannell, M. Dawson, S. L. Kelly, Cytochrome P450105D1 (CYP105D1) from Streptomyces Griseus; Heterologous Expression, Activity and Activation Effects of Multiple Xenobiotics, Biochem. Biophys. Res. Commun., Vol. 263, 1999, pp. 838-842, https://doi.org/10.1006/bbrc.1999.1427.
[19] K. P. Conner, C. M. Woods, W. M. Atkins, Interactions of Cytochrome P450s with Their Ligands, Arch. Biochem. Biophys., Vol. 507, 2011, pp. 56-65, https://doi.org/10.1016/j.abb.2010.10.00.
[20] C. Jefcoate, Measurement of Substrate and Inhibitor Binding to Microsomal Cytochrome P-450 by Optical-Difference Spectroscopy, Methods Enzymol., Vol. 1978, Elsevier, pp. 258-279,
https://doi.org/10.1016/s0076-6879(78)52029-6 .
[21] B. Dangi, K. H. Kim, S. H. Kang, T. J. Oh, Tracking Down a New Steroid-Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233-SM, Chembiochem, Vol. 19, 2018, pp. 1066-1077,
https://doi.org/10.1002/cbic.201800018.
[22] B. Dangi, C. W. Lee, K. H. Kim, S. H. Park, E. J. Yu, C. S. Jeong, H. Park, J. H. Lee, T. J. Oh, Characterization of Two Steroid Hydroxylases from Different Streptomyces spp. and Their Ligand-Bound and -Unbound Crystal Structures, FEBS J., Vol. 286, 2019, pp. 1683-1699, https://doi.org/10.1111/febs.14729.
[23] L. M. Podust, Y. Kim, M. Arase, B. A. Neely, B. J. Beck, H. Bach, D. H. Sherman, D. C. Lamb, S. L. Kelly, M. R. Waterman, The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1: A New Monooxygenase That Functionalizes Macrolide Ring Systems, J. Biol. Chem., Vol. 278, 2003, pp. 12214-12221, https://doi.org/10.1074/jbc.M212210200.
[24] J. Zhu, C. Shen, W. Zhao, X. Liu, J. Liu, B. Yu, Regio- and Stereoselective Hydroxylation of Testosterone by Cytochrome P450 from Streptomyces griseus ATCC 13273, Biocatal, Biotransformation, Vol. 39, 2021, pp. 130-137, https://doi.org/10.1080/10242422.2020.1799990.
[25] W. Y. Tong, X. Dong, Microbial Biotransformation: Recent Developments on Steroid Drugs, Recent Pat. Biotechnol., Vol. 3, 2009, pp. 141-153, https://doi.org/10.2174/187220809788700157.
[2] J. A. McIntosh, C. C. Farwell, F. H. Arnold, Expanding P450 Catalytic Reaction Space through Evolution and Engineering, Curr. Opin. Chem. Biol., Vol. 19, 2014, pp. 126-134, https://doi.org/10.1016/j.cbpa.2014.02.001.
[3] D. R. Nelson, Cytochrome P450 Diversity in the Tree of Life, Biochim, Biophys, Acta Proteins Proteomics, Vol. 1866, 2018, pp. 141-154, https://doi.org/10.1016/j.bbapap.2017.05.003.
[4] M. Moir, J. J. Danon, T. A. Reekie, M. Kassiou, An Overview of Late-Stage Functionalization in Today’s Drug Discovery, Expert Opin, Drug Discov., Vol. 14, 2019, pp. 1137-1149, https://doi.org/10.1080/17460441.2019.1653850.
[5] M. A. Cho, S. Han, Y. R. Lim, V. Kim, H. Kim, D. Kim, Streptomyces Cytochrome P450 Enzymes and Their Roles in the Biosynthesis of Macrolide Therapeutic Agents, Biomol. Ther., Vol. 27, 2019, pp. 127-133,
https://doi.org/10.4062%2Fbiomolther.2018.183.
[6] D. C. Lamb, F. P. Guengerich, S. L. Kelly, M. R. Waterman, Exploiting Streptomyces Coelicolor A3(2) P450s as a Model for Application in Drug Discovery, Expert Opin. Drug Metab. Toxicol., Vol. 2, 2006, pp. 27-40,
https://doi.org/10.1517/17425255.2.1.27.
[7] J. D. Rudolf, C. Y. Chang, M. Ma, B. Shen, Cytochromes P450 for Natural Product Biosynthesis in Streptomyces: Sequence, Structure, and Function, Nat. Prod. Rep., Vol. 34, 2017, pp. 1141-1172,
https://doi.org/10.1039/c7np00034k.
[8] A. Worsch, F. K. Eggimann, M. Girhard, C. J. von Bühler, F. Tieves, R. Czaja, A. Vogel, C. Grumaz, K. Sohn, S. Lütz, M. Kittelmann, V. B. Urlacher, A Novel Cytochrome P450 Mono-Oxygenase from Streptomyces Platensis Resembles Activities of Human Drug Metabolizing P450s, Biotechnol. Bioeng., Vol. 115, 2018, pp. 2156-2166,
https://doi.org/10.1002/bit.26781.
[9] N. Kanoh, A. K. Asano, K. Suzuki, Y. Takahashi, T. Miyazawa, T. Nakamura, T. Moriya,
H. Hirano, H. Osada, Y. Iwabuchi, S. Takahashi, An Integrated Screening System for the Selection of Exemplary Substrates for Natural and Engineered Cytochrome P450s, Sci. Rep., Vol. 9, 2019, pp. 18023, https://doi.org/10.1038/s41598-019-54473-8.
[10] H. N. T. Vu, D. T. Nguyen, H. Q. Nguyen, H. H. Chu, S. K. Chu, M. Van Chau, Q. T. Phi, Antimicrobial and Cytotoxic Properties of Bioactive Metabolites Produced by Streptomyces cavourensis YBQ59 Isolated from Cinnamomum cassia Prels in Yen Bai Province of Vietnam, Curr. Microbiol., Vol. 75, 2018, pp. 1247-1255, https://doi.org/10.1007/s00284-018-1517-x.
[11] T. Omura, R. Sato, The Carbon Monoxide-Binding Pigment of Liver Microsomes, J. Biol. Chem., Vol. 239, 1964, pp. 2370-2378, https://doi.org/10.1016/S0021-9258(20)82244-3.
[12] K. C. Leibman, A. G. Hildebrandt, R. M. Estabrook, Spectrophotometric Studies of Interactions Between Various Substrates in Their Binding to Microsomal Cytochrome P-450, Biochem. Biophys. Res. Commun., Vol. 36, 1969, pp. 789-794, https://doi.org/10.1016/0006-291X(69)90678-0.
[13] T. T. B. Ly, Y. Khatri, J. Zapp, M. C. Hutter, R. Bernhardt, CYP264B1 from Sorangium cellulosum So ce56: A Fascinating Norisoprenoid and Sesquiterpene Hydroxylase, Appl. Microbiol. Biotechnol., Vol. 95, 2012, pp. 123-133, https://doi.org/10.1007/s00253-011-3727-z.
[14] P. Subedi, K. H. Kim, Y. S. Hong, J. H. Lee, T. J. Oh, Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 from Streptomyces Species, J. Microbiol. Biotechnol., Vol. 31, 2021, pp. 464-474, https://doi.org/10.4014%2Fjmb.2010.10020.
[15] J. McCoy, E. LaVallie, Expression and Purification of Thioredoxin Fusion Proteins, Curr. Protoc. Mol. Biol., Vol. 28, 1994, pp. 16-8, https://doi.org/10.1002/0471140864.ps0607s10.
[16] T. Makino, Y. Katsuyama, T. Otomatsu, N. Misawa, Y. Ohnishi, Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces Griseus Cytochrome P450 CYP154C3, Appl, Environ. Microbiol., Vol. 80, 2013, pp. 1371-1379, https://doi.org/10.1128/aem.03504-13.
[17] M. A. Kaderbhai, C. C. Ugochukwu, S. L. Kelly, D. C. Lamb, Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia Coli, J. Appl. Environ. Microbiol., Vol. 67, 2001, pp. 2136-2138, https://doi.org/10.1128/aem.67.5.2136-2138.200.
[18] M. Taylor, D. C. Lamb, R. Cannell, M. Dawson, S. L. Kelly, Cytochrome P450105D1 (CYP105D1) from Streptomyces Griseus; Heterologous Expression, Activity and Activation Effects of Multiple Xenobiotics, Biochem. Biophys. Res. Commun., Vol. 263, 1999, pp. 838-842, https://doi.org/10.1006/bbrc.1999.1427.
[19] K. P. Conner, C. M. Woods, W. M. Atkins, Interactions of Cytochrome P450s with Their Ligands, Arch. Biochem. Biophys., Vol. 507, 2011, pp. 56-65, https://doi.org/10.1016/j.abb.2010.10.00.
[20] C. Jefcoate, Measurement of Substrate and Inhibitor Binding to Microsomal Cytochrome P-450 by Optical-Difference Spectroscopy, Methods Enzymol., Vol. 1978, Elsevier, pp. 258-279,
https://doi.org/10.1016/s0076-6879(78)52029-6 .
[21] B. Dangi, K. H. Kim, S. H. Kang, T. J. Oh, Tracking Down a New Steroid-Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233-SM, Chembiochem, Vol. 19, 2018, pp. 1066-1077,
https://doi.org/10.1002/cbic.201800018.
[22] B. Dangi, C. W. Lee, K. H. Kim, S. H. Park, E. J. Yu, C. S. Jeong, H. Park, J. H. Lee, T. J. Oh, Characterization of Two Steroid Hydroxylases from Different Streptomyces spp. and Their Ligand-Bound and -Unbound Crystal Structures, FEBS J., Vol. 286, 2019, pp. 1683-1699, https://doi.org/10.1111/febs.14729.
[23] L. M. Podust, Y. Kim, M. Arase, B. A. Neely, B. J. Beck, H. Bach, D. H. Sherman, D. C. Lamb, S. L. Kelly, M. R. Waterman, The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1: A New Monooxygenase That Functionalizes Macrolide Ring Systems, J. Biol. Chem., Vol. 278, 2003, pp. 12214-12221, https://doi.org/10.1074/jbc.M212210200.
[24] J. Zhu, C. Shen, W. Zhao, X. Liu, J. Liu, B. Yu, Regio- and Stereoselective Hydroxylation of Testosterone by Cytochrome P450 from Streptomyces griseus ATCC 13273, Biocatal, Biotransformation, Vol. 39, 2021, pp. 130-137, https://doi.org/10.1080/10242422.2020.1799990.
[25] W. Y. Tong, X. Dong, Microbial Biotransformation: Recent Developments on Steroid Drugs, Recent Pat. Biotechnol., Vol. 3, 2009, pp. 141-153, https://doi.org/10.2174/187220809788700157.