Cytochrome P450-catalyzed allylic oxidation of pentalenene to 1-deoxypentalenic acid in pentalenolactone biosynthesis

Abstract

Pentalenolactone is a sesquiterpene antibiotic from Streptomyces. Its biosynthetic pathway has been elucidated, except for the oxidation of pentalen-13-al to 1-deoxypentalenic acid. In this study, we show that cytochrome P450 pentalenene oxygenase catalyzed the formation of 1-deoxypentalenic acid. Ferredoxin XNR_5179 and ferredoxin reductase XNR_4478 from S. albus are suitable redox proteins for pentalenene oxygenase. The biosynthetic pathway presented fills a gap in the biosynthetic pathway of pentalenolactone and provides an example of cytochrome P450 enzyme activity being affected by redox proteins.

Keywords

Streptomyces / Pentalenene oxygenase / Ferredoxin / Ferredoxin reductase / Pentalenolactone

Data Availability Statement

All data generated or analysed during this study are included in this published article and its supplementary information files or are available upon request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT Authorship Contribution Statement

Jing Li: Writing - original draft, Methodology, Investigation, Formal analysis. Chengde Zhang: Methodology, Investigation. Shiwen Wu: Methodology, Investigation. Jiao Xue: Investigation, Formal analysis. Ke Chen: Investigation, Formal analysis. Zixin Deng: Supervision, Funding acquisition. Dongqing Zhu: Writing - review & editing, Supervision, Funding acquisition, Conceptualization.

Acknowledgments

This work was supported by a grant from the National Key research and development Program of China (2021YFA0909500), and National Natural Science Foundation of China (31401057).

We thank Tiangang Liu (Shanghai Jiao Tong University, Shanghai, China) for providing the E. coli strain BL21(DE3) (pMH1, pFZ81).

References

[1] M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Heme-containing oxygenases, Chem. Rev. 96 (1996) 2841-2887, doi:10.1021/Cr9500500.

[2] R. Bernhardt, Cytochromes P450 as versatile biocatalysts, J. Biotechnol. 124 (2006) 128-145, doi:10.1016/j.jbiotec.2006.01.026.

[3] V.B. Urlacher, M. Girhard, Cytochrome P450 monooxygenases in biotech- nology and synthetic biology, Trend. Biotechnol. 37 (2019) 882-897, doi:10.1016/j.tibtech.2019.01.001.

[4] D.E. Cane, T. Rossi, J.P. Pachlatko, Biosynthesis of pentalenolactone, Tetrahedron. Lett. (1979) 3639-3642.

[5] F. Pfeiffer, D. Mecke, Purification of pentalenolactone-insensitive glyceraldehy- de-3-phosphate dehydrogenase from the producer strain streptomyces arenae, H-S Z. Physiol. Chem. 361 (1980) 314.

[6] D.E. Cane, P.H.M. Harrison, C.R. Lamberson, J.S. Oliver, J.K. Sohng, Biosynthesis of pentalenolactone - isolation of the pentalenene synthase gene, Abstr. Pap. Am. Chem. S. 203 (1992) 390-ORGN.

[7] L.Y. Chen, Q. Deng, T.T. Ma, J.Y. Gu, J. Yang, X. Zhang, Y.Q. Zou, Z.X. Deng, L. Chen, C. M. Zhao, Substrate-dependent mechanism switch in the desaturation reactions of the mononuclear nonheme iron enzyme PtlD, ACS Catal. 14 (2024) 7389-7401, doi:10.1021/acscatal.4c00757.

[8] C.N. Tetzlaff, Z. You, D.E. Cane, S. Takamatsu, S. Omura, H. Ikeda, A gene cluster for biosynthesis of the sesquiterpenoid antibiotic pentalenolactone in Streptomyces avermitilis, Biochem.-Us 45 (2006) 6179-6186, doi:10.1021/bi060419n.

[9] R. Quaderer, S. Omura, H. Ikeda, D.E. Cane, Pentalenolactone biosynthesis.: molecular cloning and assignment of biochemical function to PtII, a cytochrome p450 of Streptomyces avermitilis, J. Am. Chem. Soc. 128 (2006) 13036-13037, doi:10.1021/ja0639214.

[10] Z. You, S. Omura, H. Ikeda, D.E. Cane, Pentalenolactone biosynthesis.: molec- ular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis, J. Am. Chem. Soc. 128 (2006) 6566-6567, doi:10.1021/ja061469i.

[11] Z. You, S. Omura, H. Ikeda, D.E. Cane, Pentalenolactone biosynthesis: molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate, Arch. Biochem. Biophys. 459 (2007) 233-240, doi:10.1016/j.abb.2006.11.016.

[12] Z. You, S. Omura, H. Ikeda, D.E. Cane, G. Jogl, Crystal structure of the non-heme iron dioxygenase PtIH in pentalenolactone biosynthesis, J. Biol. Chem. 282 (2007) 36552-36560, doi:10.1074/jbc.M706358200.

[13] J. Jiang, C.N. Tetzlaff, S. Takamatsu, M. Iwatsuki, M. Komatsu, H. Ikeda, D.E. Cane, Genome mining in streptomyces avermitilis: a biochemical Baeyer-Villiger reaction and discovery of a new branch of the Pentalenolactone Family tree, Biochem.-Us 48 (2009) 6431-6440, doi:10.1021/bi900766w.

[14] M.J. Seo, D.Q. Zhu, S. Endo, H. Ikeda, D.E. Cane, Genome mining in strepto- myces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone, Biochem.-Us 50 (2011) 1739-1754, doi:10.1021/bi1019786.

[15] S. Takamatsu, L.H. Xu, S. Fushinobu, H. Shoun, M. Komatsu, D.E. Cane, H. Ikeda, Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolac- tone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis, J. Antibiot. 64 (2011) 65-71, doi:10.1038/ja.2010.135.

[16] D.Q. Zhu, M.J. Seo, H. Ikeda, D.E. Cane, Genome mining in streptomyces. Discovery of an unprecedented P450-catalyzed oxidative rearrangement that is the final step in the biosynthesis of pentalenolactone, J. Am. Chem. Soc. 133 (2011) 2128-2131, doi:10.1021/ja111279h.

[17] D.Q. Zhu, Y.P. Wang, M.M. Zhang, H. Ikeda, Z.X. Deng, D.E. Cane, Product- mediated regulation of pentalenolactone biosynthesis in streptomyces species by the MarR/SlyA Family activators PenR and PntR, J. Bacteriol. 195 (2013) 1255-1266, doi:10.1128/Jb.02079-12.

[18] K. Chen, S.W. Wu, L. Zhu, C.D. Zhang, W.S. Xiang, Z.X. Deng, H. Ikeda, D.E. Cane, D. Q. Zhu, Substitution of a single amino acid reverses the regiospecificity of the Baeyer-Villiger monooxygenase PntE in the biosynthesis of the antibiotic pentaleno- lactone, Biochem.-Us 55 (2016) 6696-6704, doi:10.1021/acs.biochem.6b01040.

[19] L. Duan, G. Jogl, D.E. Cane, The cytochrome P450-catalyzed oxidative rear- rangement in the final step of pentalenolactone biosynthesis: substrate struc- ture determines mechanism, J. Am. Chem. Soc. 138 (2016) 12678-12689, doi:10.1021/jacs.6b08610.

[20] X.Y. Wang, J.Y. Shi, Y.J. Liu, Oxidative rearrangement mechanism of Pentalenolac- tone F catalyzed by cytochrome P450 CYP161C2 (PntM), Inorg. Chem. 57 (2018) 8933-8941, doi:10.1021/acs.inorgchem.8b00860.

[21] S. Chen, X.Z. Mao, Y.L. Shen, Y.J. Zhou, J.L. Li, L.R. Wang, X.Y. Tao, L. Yang, Y. X. Wang, X.F. Zhou, Z.X. Deng, D.Z. Wei, Tailoring the P450 monooxygenase gene for FR-008/candicidin biosynthesis, Appl. Environ. Microb. 75 (2009) 1778-1781, doi:10.1128/Aem.00859-08.

[22] M. Carmody, B. Murphy, B. Byrne, P. Power, D. Rai, B. Rawlings, P. Caffrey, Biosyn- thesis of amphotericin derivatives lacking exocyclic carboxyl groups, J. Biol. Chem. 280 (2005) 34420-34426, doi:10.1074/jbc.M506689200.

[23] T. Brautaset, H. Sletta, A. Nedal, S.E.F. Borgos, K.F. Degnes, I. Bakke, O. Volokhan, O.N. Sekurova, I.D. Treshalin, E.P. Mirchink, A. Dikiy, T.E. Ellingsen, S.B. Zotchev, Improved antifungal polyene macrolides via engineering of the nys- tatin biosynthetic genes in streptomyces noursei, Chem. Biol. 15 (2008) 1198-1206, doi:10.1016/j.chembiol.2008.08.009.

[24] Z. Qi, Q.J. Kang, C.Y. Jiang, M. Han, L.Q. Bai, Engineered biosynthesis of pimaricin derivatives with improved antifungal activity and reduced cytotoxicity, Appl. Mi- crobiol. Biot. 99 (2015) 6745-6752, doi:10.1007/s00253-015-6635-9.

[25] Y. Iizaka, R. Takeda, Y. Senzaki, A. Fukumoto, Y. Anzai, Cytochrome P450 enzyme RosC catalyzes a multistep oxidation reaction to form the non-active compound 20-carboxyrosamicin, Fem. Microbiol. Lett. 364 (2017), doi:10.1093/femsle/fnx110.

[26] J.W. Zhou, T.Y. Hu, Y. Liu, L.C. Tu, Y.D. Song, Y. Lu, Y.F. Zhang, Y.R. Tong, Y.J. Zhao, P. Su, X.Y. Wu, L.Q. Huang, W. Gao, Cytochrome P450 cataly- ses the 29-carboxyl group formation of celastrol, Phytochemistry 190 (2021), doi:10.1016/j.phytochem.2021.112868.

[27] C.A. Helliwell, A. Poole, W.J. Peacock, E.S. Dennis, Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis, Plant. Physiol. 119 (1999) 507-510, doi:10.1104/Pp.119.2.507.

[28] C.A. Helliwell, P.M. Chandler, A. Poole, E.S. Dennis, W.J. Peacock, The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gib- berellin biosynthesis pathway, P. Natl. Acad. Sci. U.S.A. 98 (2001) 2065-2070, doi:10.1073/pnas.041588998.

[29] I.A. Pikuleva, A. Babiker, M.R. Waterman, I. Bjorkhem, Activities of recombi- nant human cytochrome P450c27 (CYP27) which produce intermediates of alter- native bile acid biosynthetic pathways, J. Biol. Chem. 273 (1998) 18153-18160, doi:10.1074/jbc.273.29.18153.

[30] D.K. Ro, E.M. Paradise, M. Ouellet, K.J. Fisher, K.L. Newman, J.M. Ndungu, K.A. Ho, R.A. Eachus, T.S. Ham, J. Kirby, M.C.Y. Chang, S.T. Withers, Y. Shiba, R. Sarpong, J. D. Keasling, Production of the antimalarial drug precursor artemisinic acid in en- gineered yeast, Nature 440 (2006) 940-943, doi:10.1038/nature04640.

[31] K.H. Teoh, D.R. Polichuk, D.W. Reed, G. Nowak, P.S. Covello, Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin, Feb. Lett. 580 (2006) 1411-1416, doi:10.1016/j.febslet.2006.01.065.

[32] K. Yoneyama, N. Mori, T. Sato, A. Yoda, X.N. Xie, M. Okamoto, M. Iwanaga, T. Ohnishi, H. Nishiwaki, T. Asami, T. Yokota, K. Akiyama, K. Yoneyama, T. No- mura, Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis, New Phytol. 218 (2018) 1522-1533, doi:10.1111/nph.15055.

[33] F.Y. Zhu, X.F. Zhong, M.Z. Hu, L. Lu, Z.X. Deng, T.G. Liu, In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli, Biotechnol. Bioeng. 111 (2014) 1396-1405, doi:10.1002/bit.25198.

[34] Y.J. Chun, T. Shimada, R. Sanchez-Ponce, M.R. Waterman, F.P. Guengerich, Elec- tron transport pathway for a streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in streptomyces coelicolor A3(2), Drug. Metab. Rev. 39 (2007) 196.

[35] K.M. Ewen, F. Hannemann, Y. Khatri, O. Perlova, R. Kappl, D. Krug, J. Hütter- mann, R. Müller, R. Bernhardt, Genome mining in sorangium cellulosum so ce56 identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450, J. Biol. Chem. 284 (2009) 28590-28598, doi:10.1074/jbc.M109.021717.

[36] B.P. Pandey, N. Lee, K.Y. Choi, J.N. Kim, E.J. Kim, B.G. Kim, Identification of the spe- cific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680, Appl. Microbiol. Biot. 98 (2014) 5009-5017, doi:10.1007/s00253-014-5525-x.

[37] W. Zhang, Y. Liu, J.Y. Yan, S.N. Cao, F.L. Bai, Y. Yang, S.H. Huang, L.S. Yao, Y. Anzai, F. Kato, L.M. Podust, D.H. Sherman, S.Y. Li, New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners, J. Am. Chem. Soc. 136 (2014) 3640-3646, doi:10.1021/ja4130302.

[38] S.Y. Li, L. Du, R. Bernhardt, Redox partners: function modulators of bacterial P450 enzymes, Trend. Microbiol. 28 (2020) 445-454, doi:10.1016/j.tim.2020.02.012.