Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns

Abstract

Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from Bacillus megaterium ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners. We found that electron transfer protein 1(516–618) showed the highest yield of the main product, 15β-hydroxyprogesterone, and, furthermore, produced a reduced amount of unwanted polyhydroxylated side products. Molecular protein–protein docking indicated that this is caused by subtle structural changes leading to alternative binding modes of both redox enzymes. Stopped-flow measurements analyzing the CYP106A2 reduction and showing substantial differences in the apparent rate constants supported this conclusion. The study provides for the first time to our knowledge rational explanations for differences in product patterns of a cytochrome P450 caused by difference in the binding mode of the redox partners.

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding

“…Obviously, spacial variations in the used ferredoxins, that is, Etp1(516–618) and Adx(4–108), influence binding, and thus electron transfer, to the P450. As shown by molecular docking investigations , certain amino acids of Adx(4–108) prevent it from binding to CYP106A2 as closely as Etp1(516–618). From this observation the question arose whether mutations of the relevant amino acids in Adx(4–108) that make this region more similar to Etp1(516–618), will change the selectivity of progesterone conversion by CYP106A2 toward a higher yield of the main product, 15β‐OH‐progesterone.…”

“…Selectivity and product distribution of steroid conversions by bacterial P450s is not only dependent on properties of amino acids in the active site of the protein but also dependent on the redox partners, which can drastically influence the amount of polyhydroxylated products being formed . Obviously, spacial variations in the used ferredoxins, that is, Etp1(516–618) and Adx(4–108), influence binding, and thus electron transfer, to the P450.…”

“…These mutants did not show substantial changes compared to wild‐type Adx of their V max values of CYP11A1‐ and CYP11B1‐dependent activities, but showed variations in K m leading to the suggestion that mutants of Y82 directly or indirectly (by inducing small conformational changes) affect binding of Adx to cytochromes P450. Further considerations regarding the choice of mutations came from our previous results of protein–protein docking of Etp1(516–618) to CYP106A2 . For Etp1, we found that the side chain of phenylalanine that corresponds to Tyr82 in adrenodoxin deeply penetrates into CYP106A2 by forming hydrophobic contacts with Phe107, Pro359, and Leu356.…”

“…The above mentioned differences seen in the product spectra upon the conversion of progesterone by Adx(4–108) and Etp1(516–618) as electron donors to CYP106A2 were thus explained by subtle changes in amino acids between Etp1 and Adx that lead to different binding modes to CYP106A2 . The results from protein–protein docking studies showed a substantial increase in the FeS cluster/heme distance when applying Adx(4–108) instead of Etp1(516–618).…”

“…Likewise, the choice of the redox partner – the ferredoxin, which delivers the electrons – influences the amount and kind of products being formed. As shown before, the enzymatic conversion of progesterone using bovine adrenodoxin Adx(4–108) as an electron donor for CYP106A2 produces, besides the main product 15β‐hydroxyprogesterone (15β‐OH‐P), several monohydroxylated side products as well as polyhydroxylated progesterone . In contrast, the adrenodoxin‐like electron transfer protein 1 [Etp1(516–618)] from Schizosaccharomyces pombe , which is homologous both in sequence and structure to bovine adrenodoxin, surprisingly was found to produce a substantially lower amount of side products .…”