Abstract
Cytochrome P450 enzyme CYP109B1 is a versatile biocatalyst exhibiting hydroxylation activities toward various substrates. However, the regio- and stereoselective steroid hydroxylation by CYP109B1 is far less explored. In this study, the oxidizing activity of CYP109B1 is reconstituted by coupling redox pairs from different sources, or by fusing it to the reductase domain of two self-sufficient P450 enzymes P450RhF and P450BM3 to generate the fused enzyme. The recombinant Escherichia coli expressing necessary proteins are individually constructed and compared in steroid hydroxylation. The ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499) from Synechococcus elongates is found to be the best redox pair for CYP109B1, which gives above 99% conversion with 73% 15β selectivity for testosterone. By contrast, the rest ones and the fused enzymes show much less or negligible activity. With the aid of redox pair of Fdr_0978/Fdx_1499, CYP109B1 is used for hydroxylating different steroids. The results show that CYP109B1 displayed good to excellent activity and selectivity toward four testosterone derivatives, giving all 15β-hydroxylated steroids as main products except for 9 (10)-dehydronandrolone, for which the selectivity is shifted to 16β. While for substrates bearing bulky substitutions at C17 position, the activity is essentially lost. Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is revealed by computational analysis, thus providing theoretical basis for directed evolution to further improve its catalytic properties.
Highlights
Cytochrome P450 monooxygenases (CYPs) are heme-containing monooxygenase enzymes, which are broadly distributed among biological kingdoms and extensively involved in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, drug metabolism etc. (Munro et al, 2007; Bernhardt and Urlacher, 2014; Li et al, 2020)
Full length gene of CYP109B1 was amplified from the genome of Bacillus subtilis 168 (Kunst et al, 1997; Zhang et al, 2020) using specific upstream and downstream primers by polymerase chain reaction (PCR) (Supplementary Table S1), the resulted PCR fragment was ligated into the expression vector pRSFDuet-1 under control of T7 promoter using T5 exonuclease-dependent assembly approach (Xia et al, 2019)
The recombinant expression of P450 CYP109B1 was performed in E. coli (BL21) as a soluble protein
Summary
Cytochrome P450 monooxygenases (CYPs) are heme-containing monooxygenase enzymes, which are broadly distributed among biological kingdoms and extensively involved in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, drug metabolism etc. (Munro et al, 2007; Bernhardt and Urlacher, 2014; Li et al, 2020). (Munro et al, 2007; Bernhardt and Urlacher, 2014; Li et al, 2020) They are considered to be the most versatile biocatalysts with the capability of catalyzing functionalization of non-activated hydrocarbons under. Microbial CYPs shows great superiority over the eukaryotic ones (Donova and Egorova, 2012; Li et al, 2020a). Because they can be overexpressed in high amounts in soluble form (Bernhardt and Urlacher, 2014) and are generally much more active with turnover numbers go from ten to a few hundred molecules per min. Most of them have been employed and subjected to directed evolution to improve the both activity and selectivity for steroid hyxroxylation
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