Exploring the Potential of Cytochrome P450 CYP109B1 Catalyzed Regio-and Stereoselective Steroid Hydroxylation

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.

Keywords: CYP109B1; cytochrome P450; redox partner; regioselectivity; stereoselectivity; steroids hydroxylation.

FIGURE 1

(A): The configuration of plasmids containing CYP109B1 with different pairs of redox partners (ferredoxin and ferredoxin reductase) or plasmids containing fused enzyme by fusing the heme domain of CYP109B1 to reductase domain of P450 BM3 or P450RhF. a: plasmid pRSFDuet-1 containing CYP109B1 and Fdr_0978/Fdx_1499 redox partner, b: plasmid pRSFDuet-1 containing CYP109B1 and FNR/Fd I redox partner from spinach, c: plasmid pRSFDuet-1 containing CYP109B1 and Fpr/YkuN, d: plasmid pRSFDuet-1 containing CYP109B1 and Fpr/YkuP, e: plasmid pRSFDuet-1 containing fused enzyme P450CYP109B1-BM3 in which heme domain of CYP109B1 was fused to the reductase domain of P450 BM3 from Bacillus megaterium, f: plasmid pRSFDuet-1 containing fused enzyme P450CYP109B1-RhFRed in which heme domain of CYP109B1 was fused to the reductase domain of P450RhF from Rhodococcus sp. Strain NCIMB 9784. (B): SDS-PAGE analysis of recombination E. coli cells containing the corresponding plasmids in Figure 1A. (C): Conversion of testosterone with whole-cell catalyst expressing different reconstituted catalytic system. Reaction conditions: recombinant E. coli cells frozen with liquid nitrogen was suspended in phosphate buffer (pH 8.0, 100 mM, OD₆₀₀ =20) containing 1 unit glucose-6-phosphate dehydrogenase, 5 g/L glucose-6-phosphate, 5 g/L glycerol and 1 mM NADP⁺, reactions were conducted at 25°C, 200 rpm for 20 h.

FIGURE 2

Regio- and stereoselective hydroxylation with CYP109B for eight different steroid substrates: testosterone (1), nandrolone (2), boldenone (3), 9 (10)-dehydronandrolone (4), progesterone (5), Canreone (6), Prednisolone (7) and Pregnenolone (8). Whole cell reaction of CYP109B1-Fdr_Fdx displayed catalyze activity for substrate 1 to 4, and major products were listed.

FIGURE 3

The docked conformation of steroid substrates in the active pocket of CYP109B1 (PDB ID: 4RM4). (A): Testosterone. (B): Nandrolone. (C): Boldenone. (D): 9 (10) dehydronandrolone .Key residues in active pocket of CYP109B1 are colored in violet and residues forming hydrogen bonds are labeled in blue. The important distance in the favorable docked poses are given in angstrom (Å). The C15 atom of steroid substrates are displayed in green with ball style and the Ox atom of Cpd I are displayed in red with ball style.

FIGURE 4

The fluctuation of the distance of Fe—O3 (red), Fe—C15 (orange) and Fe—C16 (blue) in CYP109B1-Progesterone systems (A) and CYP109B1-Progesterone systems (B) during the MD simulations.