Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469)

Qiuping  Yao; Li  Ma; Ling  Liu; Haruo  Ikeda; Shinya  Fushinobu; Shengying  Li; Lian-Hua  Xu

doi:10.4014/jmb.1610.10079

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Research article

Biotechnology and Bioengineering (BB) | Enzyme, Protein Engineering, and Metabolic Engineering

Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469)

Qiuping Yao ¹, Li Ma ², Ling Liu ¹, Haruo Ikeda ³, Shinya Fushinobu ⁴, Shengying Li ² and Lian-Hua Xu ^1*

¹Ocean College, Zhejiang University, Dinghai, Zhoushan, Zhejiang 316021, P.R. China, ²Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, P.R. China, ³Kitasato Institute for Life Sciences, Kitasato University, Kanagawa 252-0373, Japan, ⁴Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

Received: November 1, 2016; Accepted: March 9, 2017

J. Microbiol. Biotechnol. 2017; 27(5): 956-964

Published May 28, 2017 https://doi.org/10.4014/jmb.1610.10079

Abstract

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steadystate kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The Km and kcat values for compactin were 39.1 ± 8.8 μM and 1.12 ± 0.09 min-1, respectively. The kcat/Km value for compactin (0.029 min-1·μM-1) was lower than that for diclofenac (0.114 min-1·μM-1). Spectroscopic analysis showed that CYP105D7 binds to compactin with a Kd value of 17.5 ± 3.6 μM. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

Keywords

Compactin, CYP105D7, pravastatin, Streptomyces avermitilis, hydroxylation

References

Xu LH, Fushinobu S, Ikeda H, Wakagi T, Shoun H. 2009. Crystal structures of cytochrome P450 105P1 from Streptomyces avermitilis: conformational flexibility and histidine ligation state. J. Bacteriol. 191: 1211-1219.
Makino T, Katsuyama Y, Otomatsu T, Misawa N, Ohnishi Y. 2014. 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. 80: 1371-1379.
Podust LM, Sherman DH. 2012. Diversity of P450 enzymes in the biosynthesis of natural products. Nat. Prod. Rep. 29:1251-1266.
Bernhardt R. 2006. Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124: 128-145.
Ma L, Du L, Chen H, Sun Y, Huang S, Zheng X, et al. 2015. Reconstitution of the in vitro activity of the cyclosporinespecific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system. Appl. Environ. Microbiol. 81: 6268-6275.
Fasan R. 2012. Tuning P450 enzymes as oxidation catalysts. ACS Catal. 2: 647-666.
Kelly SL, K elly DE, J ackson C J, W arrilow A G, L amb DC. 2005. The diversity and importance of microbial cytochromes P450, pp. 585-617. In Ortiz de Montellano PR (ed.). Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd Ed. Kluwer Academic/Plenum Publishers, New York. USA.
Moody SC, Loveridge E J. 2 014. C YP105 — diverse structures, functions and roles in an intriguing family of enzymes in Streptomyces. J. Appl. Microbiol. 117: 1549-1563.
Taylor M, Lamb DC, Cannell R, Dawson M, Kelly SL. 1999. Cytochrome P450105D1 (CYP105D1) from Streptomyces griseus:heterologous expression, activity, and activation effects of multiple xenobiotics. Biochem. Biophys. Res. Commun. 263:838-842.
Chun Y-J, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao B, et al. 2007. Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2). J. Biol. Chem. 282: 17486-17500.
Xu L-H, Fushinobu S, Takamatsu S, Wakagi T, Ikeda H, Shoun H. 2010. Regio- and stereospecificity of filipin hydroxylation sites revealed by crystal structures of cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis. J. Biol. Chem. 285: 16844-16853.
Takamatsu S, Xu L-H, Fushinobu S, Shoun H, Komatsu M, Cane DE, et al. 2011. Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites:hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis. J. Antibiot. 64: 65-71.
Xu L-H, Ikeda H, Liu L, Arakawa T, Wakagi T, Shoun H, Fushinobu S. 2015. Structural basis for the 4’-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase. Appl. Microbiol. Biotechnol. 99: 3081-3091.
Liu L, Yao Q, Ma Z, Ikeda H, Fushinobu S, Xu L-H. 2016. Hydroxylation of flavanones by cytochrome P450 105D7 from Streptomyces avermitilis. J. Mol. Catal. B Enzym. 132: 91-97.
Zhang J, Lu X, Li J-J. 2013. Conversion of fatty aldehydes into alk(a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system. Biotechnol. Biofuels 6: 1.
Endo A, Kuroda M, Tsujita Y. 1976. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J. Antibiot. 29: 1346-1348.
Serizawa N. 1996. Biochemical and molecular approaches for production of pravastatin, a potent cholesterol-lowering drug. Biotechnol. Annu. Rev. 2: 373-389.
Matsuoka T, Miyakoshi S, Tanzawa K, Nakahara K, Hosobuchi M, Serizawa N. 1989. Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. Eur. J. Biochem. 184: 707-713.
McLean KJ, Hans M, Meijrink B, van Scheppingen WB, Vollebregt A, Tee KL, et al. 2015. Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proc. Natl. Acad. Sci. USA 112: 2847-2852.
Urlacher VB, Girhard M. 2012. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30: 26-36.
Guengerich FP. 2002. Rate-limiting steps in cytochrome P450 catalysis. Biol. Chem. 383: 1553-1564.
Watanabe I, Nara F, Serizawa N. 1995. Cloning, characterization and expression of the gene encoding cytochrome P-450sca-in2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene 163: 81-85.
Park J-W, Lee J-K, Kwon T-J, Yi D-H, Kim Y-J, Moon S-H, et al. 2003. Bioconversion of compactin into pravastatin by Streptomyces sp. Biotechnol. Lett. 25: 1827-1831.
Peng Y, Demain AL. 2000. Bioconversion of compactin to pravastatin by Actinomadura sp. ATCC 55678. J. Mol. Catal. B Enzym. 10: 151-156.
Chen C-H, Hu H-Y, Cho Y-C, Hsu W-H. 2006. Screening of compactin-resistant microorganisms capable of converting compactin to pravastatin. Curr. Microbiol. 53: 108-112.
Fujii Y, Norihisa K, Fujii T, Aritoku Y, Kagawa Y, Sallam KI, et al. 2011. Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor. Biochem. Biophys. Res. Commun. 404: 511-516.
Wester MR, Johnson EF, Marques-Soares C, Dijols S, Dansette PM, Mansuy D, Stout CD. 2003. Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1Å resolution: evidence for an induced fit model of substrate binding. Biochemistry 42: 9335-9345.
Ba L, Li P, Zhang H, Duan Y, Lin Z. 2013. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli. Biotechnol. J. 8: 785-793.
Ba L, Li P, Zhang H, Duan Y, Lin Z. 2013. Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer. Biotechnol. Bioeng. 110: 2815-2825.
Pandey BP, Roh C, Choi KY, Lee N, Kim EJ, Ko S, et al. 2010. Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680. Biotechnol. Bioeng. 105: 697-704.
Li S, Chaulagain MR, Knauff AR, Podust LM, Montgomery J, Sherman DH. 2009. Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase. Proc. Natl. Acad. Sci. USA 106: 18463-18468.
Zhang W, Liu Y, Yan J, Cao S, Bai F, Yang Y, et al. 2014. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners. J. Am. Chem. Soc. 136: 3640-3646.
Bernhardt R , Urlacher V B. 2 014. C y tochromes P450 a s promising catalysts for biotechnological application: chances and limitations. Appl. Microbiol. Biotechnol. 98: 6185-6203.

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(5): 956-964

Published online May 28, 2017 https://doi.org/10.4014/jmb.1610.10079

Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469)

Qiuping Yao ¹, Li Ma ², Ling Liu ¹, Haruo Ikeda ³, Shinya Fushinobu ⁴, Shengying Li ² and Lian-Hua Xu ^1*

¹Ocean College, Zhejiang University, Dinghai, Zhoushan, Zhejiang 316021, P.R. China, ²Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, P.R. China, ³Kitasato Institute for Life Sciences, Kitasato University, Kanagawa 252-0373, Japan, ⁴Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

Received: November 1, 2016; Accepted: March 9, 2017

Abstract

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-
methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows
superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450,
CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic
acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze
compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox
partner systems with an NADPH regeneration system, the reaction produced two
hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steadystate
kinetic parameters were measured using the redox partners of putidaredoxin and its
reductase. The Km and kcat values for compactin were 39.1 ± 8.8 μM and 1.12 ± 0.09 min-1,
respectively. The kcat/Km value for compactin (0.029 min-1·μM-1) was lower than that for
diclofenac (0.114 min-1·μM-1). Spectroscopic analysis showed that CYP105D7 binds to
compactin with a Kd value of 17.5 ± 3.6 μM. Molecular docking analysis was performed to
build a possible binding model of compactin. Comparisons of different substrates with
CYP105D7 were conclusively illustrated for the first time.

Keywords: Compactin, CYP105D7, pravastatin, Streptomyces avermitilis, hydroxylation

Abstract

References

Xu LH, Fushinobu S, Ikeda H, Wakagi T, Shoun H. 2009. Crystal structures of cytochrome P450 105P1 from Streptomyces avermitilis: conformational flexibility and histidine ligation state. J. Bacteriol. 191: 1211-1219.
Makino T, Katsuyama Y, Otomatsu T, Misawa N, Ohnishi Y. 2014. 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. 80: 1371-1379.
Podust LM, Sherman DH. 2012. Diversity of P450 enzymes in the biosynthesis of natural products. Nat. Prod. Rep. 29:1251-1266.
Bernhardt R. 2006. Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124: 128-145.
Ma L, Du L, Chen H, Sun Y, Huang S, Zheng X, et al. 2015. Reconstitution of the in vitro activity of the cyclosporinespecific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system. Appl. Environ. Microbiol. 81: 6268-6275.
Fasan R. 2012. Tuning P450 enzymes as oxidation catalysts. ACS Catal. 2: 647-666.
Kelly SL, K elly DE, J ackson C J, W arrilow A G, L amb DC. 2005. The diversity and importance of microbial cytochromes P450, pp. 585-617. In Ortiz de Montellano PR (ed.). Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd Ed. Kluwer Academic/Plenum Publishers, New York. USA.
Moody SC, Loveridge E J. 2 014. C YP105 — diverse structures, functions and roles in an intriguing family of enzymes in Streptomyces. J. Appl. Microbiol. 117: 1549-1563.
Taylor M, Lamb DC, Cannell R, Dawson M, Kelly SL. 1999. Cytochrome P450105D1 (CYP105D1) from Streptomyces griseus:heterologous expression, activity, and activation effects of multiple xenobiotics. Biochem. Biophys. Res. Commun. 263:838-842.
Chun Y-J, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao B, et al. 2007. Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2). J. Biol. Chem. 282: 17486-17500.
Xu L-H, Fushinobu S, Takamatsu S, Wakagi T, Ikeda H, Shoun H. 2010. Regio- and stereospecificity of filipin hydroxylation sites revealed by crystal structures of cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis. J. Biol. Chem. 285: 16844-16853.
Takamatsu S, Xu L-H, Fushinobu S, Shoun H, Komatsu M, Cane DE, et al. 2011. Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites:hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis. J. Antibiot. 64: 65-71.
Xu L-H, Ikeda H, Liu L, Arakawa T, Wakagi T, Shoun H, Fushinobu S. 2015. Structural basis for the 4’-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase. Appl. Microbiol. Biotechnol. 99: 3081-3091.
Liu L, Yao Q, Ma Z, Ikeda H, Fushinobu S, Xu L-H. 2016. Hydroxylation of flavanones by cytochrome P450 105D7 from Streptomyces avermitilis. J. Mol. Catal. B Enzym. 132: 91-97.
Zhang J, Lu X, Li J-J. 2013. Conversion of fatty aldehydes into alk(a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system. Biotechnol. Biofuels 6: 1.
Endo A, Kuroda M, Tsujita Y. 1976. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J. Antibiot. 29: 1346-1348.
Serizawa N. 1996. Biochemical and molecular approaches for production of pravastatin, a potent cholesterol-lowering drug. Biotechnol. Annu. Rev. 2: 373-389.
Matsuoka T, Miyakoshi S, Tanzawa K, Nakahara K, Hosobuchi M, Serizawa N. 1989. Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. Eur. J. Biochem. 184: 707-713.
McLean KJ, Hans M, Meijrink B, van Scheppingen WB, Vollebregt A, Tee KL, et al. 2015. Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proc. Natl. Acad. Sci. USA 112: 2847-2852.
Urlacher VB, Girhard M. 2012. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30: 26-36.
Guengerich FP. 2002. Rate-limiting steps in cytochrome P450 catalysis. Biol. Chem. 383: 1553-1564.
Watanabe I, Nara F, Serizawa N. 1995. Cloning, characterization and expression of the gene encoding cytochrome P-450sca-in2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene 163: 81-85.
Park J-W, Lee J-K, Kwon T-J, Yi D-H, Kim Y-J, Moon S-H, et al. 2003. Bioconversion of compactin into pravastatin by Streptomyces sp. Biotechnol. Lett. 25: 1827-1831.
Peng Y, Demain AL. 2000. Bioconversion of compactin to pravastatin by Actinomadura sp. ATCC 55678. J. Mol. Catal. B Enzym. 10: 151-156.
Chen C-H, Hu H-Y, Cho Y-C, Hsu W-H. 2006. Screening of compactin-resistant microorganisms capable of converting compactin to pravastatin. Curr. Microbiol. 53: 108-112.
Fujii Y, Norihisa K, Fujii T, Aritoku Y, Kagawa Y, Sallam KI, et al. 2011. Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor. Biochem. Biophys. Res. Commun. 404: 511-516.
Wester MR, Johnson EF, Marques-Soares C, Dijols S, Dansette PM, Mansuy D, Stout CD. 2003. Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1Å resolution: evidence for an induced fit model of substrate binding. Biochemistry 42: 9335-9345.
Ba L, Li P, Zhang H, Duan Y, Lin Z. 2013. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli. Biotechnol. J. 8: 785-793.
Ba L, Li P, Zhang H, Duan Y, Lin Z. 2013. Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer. Biotechnol. Bioeng. 110: 2815-2825.
Pandey BP, Roh C, Choi KY, Lee N, Kim EJ, Ko S, et al. 2010. Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680. Biotechnol. Bioeng. 105: 697-704.
Li S, Chaulagain MR, Knauff AR, Podust LM, Montgomery J, Sherman DH. 2009. Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase. Proc. Natl. Acad. Sci. USA 106: 18463-18468.
Zhang W, Liu Y, Yan J, Cao S, Bai F, Yang Y, et al. 2014. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners. J. Am. Chem. Soc. 136: 3640-3646.
Bernhardt R , Urlacher V B. 2 014. C y tochromes P450 a s promising catalysts for biotechnological application: chances and limitations. Appl. Microbiol. Biotechnol. 98: 6185-6203.