Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium – identification of a novel 11-oxidase activity

Abstract

The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.

Identification of new substrates for the CYP106A1‐mediated 11‐oxidation and investigation of the reaction mechanism

“…Likewise, product P3, which was identified as a 2:3 mixture of 15β‐hydroxyprednisone and 1,2‐dihydro‐15β‐hydroxyprednisone displaying 11‐oxidation catalyzed by CYP106A1 (Scheme 1 ). The unusual 1(2)‐double bond hydrogenation has not been observed in our previous studies [22], hence, it was proposed to be the result of an uncharacterized enzyme present in the B. megaterium MS941 strain. It has been reported that 3‐ketosteroid‐dehydrogenases present in Nocardia , Mycobacterium and Actinobacter sp.…”

“…Since the previously identified substrates for the CYP106A1 11‐oxidation (corticosterone and cortisol) gave rise to multiple product formation [22], other 11β‐hydroxysteroid analogs were investigated to identify a selective 11‐keto product formation. The in vitro conversion of the pharmaceutically relevant glucocorticoids, prednisolone and dexamethasone, also resulted in several products (Table 1 , Figs.…”

“…Prednisolone, dexamethasone and 11‐OH‐AD were converted with a reconstituted system containing the CYP106A1 enzyme, AdR and Adx 4–108 (in a ratio of 1:2:20), and a NADPH regenerating system, as formerly described by Kiss et al [22]. The reaction was performed at 30 °C, for 60 min using 200 μM substrate.…”

“…Therefore, further 11β‐hydroxysteroid analogs were screened to obtain a regio‐selective C11‐oxidation. The chosen steroids, prednisolone, dexamethasone and 11β‐hydroxyandrostenedione (11‐OH‐AD), were previously identified as high‐spin‐shift‐inducing CYP106A1 substrates, yet, the reactions were not investigated in detail and the products were not characterized [22]. At first, we performed in vitro transformation of the substrates, followed by their in vivo turnover using a CYP106A1‐based B. megaterium whole‐cell system to obtain higher product yields required for the structural elucidation by nuclear magnetic resonance (NMR).…”

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

“…CYPs such as CYP106A1, CYP106A2, CYP109B1, CYP109E1, CYP154C3, CYP154C5, CYP260A1, and CYP260B1, originating from bacterial sources, are known to hydroxylate steroids . CYP154C8 shows high similarity with CYP154C3 (74 %) and CYP154C5 (66 %), both of which are reported to hydroxylate steroids at C16α.…”