Abstract
A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17β-estradiol (E2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E2 in this study. Some of the tested mutants could regioselectively generate 2-OH E2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Caswell JM, O’Neill M, Taylor SJ, Moody TS (2013) Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Curr Opin Chem Biol 17:271–275
Holl HL (1999) Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts. Steroids 64:178–186
Kang JY, Kim SY, Kim D, Kim DH, Shin SM, Park SH, Kim KH, Jung HC, Pan JG, Joung YH, Chi YT, Chae HZ, Ahn T, Yun CH (2011) Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium. AMB Express 1:1
Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol Bioeng 111:1313–1322
Kille S, Zilly FE, Acevedo JP, Reetz MT (2011) Regio-and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nat Chem 3:738–743
Kim DH, Kim KH, Kim DH, Liu KH, Jung HC, Pan JG, Yun CH (2008) Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3. Drug Metab Dispos 36:2166–2170
Lee GY, Kim DH, Kim D, Ahn T, Yun CH (2014) Functional characterization of steroid hydroxylase CYP106A1 derived from Bacillus megaterium. Arch Pharm Res. doi:10.1007/s12272-014-0366-9
Mahato SB, Gari S (1997) Advances in microbial steroid biotransformation. Steroids 62:332–345
Park SH, Kim DH, Kim D, Kim DH, Jung HC, Pan JG, Ahn T, Kim D, Yun CH (2010) Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation. Drug Metab Dispos 38:732–739
Rea V, Kolkman AJ, Vottero E, Stronks EJ, Ampt KA, Honing M, Vermeulen NP, Wijmenga SS, Commandeur JN (2012) Active site substitution A82 W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies. Biochemistry 51:750–760
Shimada T, Wunsch RM, Hanna IH, Sutter TR, Guengerich FP, Gillam EM (1998) Recombinant human cytochrome P450 1B1 expression in Escherichia coli. Arch Biochem Biophys 357:111–120
Turan VK, Sanchez RI, Li JJ, Li SA, Reuhl KR, Thomas PE, Conney AH, Gallo MA, Kauffman FC, Mesia-Vela S (2004) The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone. J Endocrinol 183:91–99
Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36
Virus C, Bernhardt R (2008) Molecular evolution of a steroid hydroxylating cytochrome P450 using a versatile steroid detection system for screening. Lipids 43:1133–1141
Whitehouse CJ, Bell SG, Wong LL (2012) P450BM3 (CYP102A1): connecting the dots. Chem Soc Rev 41:1218–1260
Yun CH, Kim KH, Kim DH, Jung HC, Pan JG (2007) The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities. Trends Biotechnol 25:289–298
Zehentgruber D, Hannemann F, Bleif S, Bernhardt R, Lütz S (2010) Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2. Chem BioChem 11:713–721
Acknowledgments
This research was supported by the Next-Generation BioGreen 21 program (SSAC, grant#: PJ00948303), Rural Development Administration, Republic of Korea, and the the Bio-industry Technology Development Program (111,052-04-3-SB010), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Conflict of interest
The authors declare no conflict of interests.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Cha, G.S., Ryu, S.H., Ahn, T. et al. Regioselective hydroxylation of 17β-estradiol by mutants of CYP102A1 from Bacillus megaterium . Biotechnol Lett 36, 2501–2506 (2014). https://doi.org/10.1007/s10529-014-1628-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-014-1628-9