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

Abstract

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4–108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4–108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4–108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein–protein docking, as well as conversion studies, using the mutants demonstrated that the iron–sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5‐fold increase in 15β‐hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

“…Such effect is not unusual for the CYP enzyme complex, as recently described for the CYP1A1 enzyme, in which substrate binding was accommodated by minimal perturbation of the α-carbon backbone with subtle adjustments of the side chains (Bart and Scott, 2018). Additionally, a single amino acid change in a redox partner of CYP106A2 induced a considerable increase in substrate conversion by this bacterial CYP, with a minimal conformational perturbation (Sagadin et al, 2019).…”

Cytochrome P450 Monooxygenases Catalyse Steroid Nucleus Hydroxylation with Regio‐ and Stereo‐Selectivity

“…The productivities of 1 and 3 was increased up to 18‐fold to yield an absolute productivity up to 5.5 g/L per day [63] (Table 1). In an alternative approach, Adx was optimized by mutation, the distance of the iron‐sulfur cluster and haem diminished significantly, leading to an approximately 2.5‐fold increase in the production of 15β‐hydroxyprogesterone [64] . The stability and activity of CYP106A2 with 24 were also tested under process conditions using the enzyme overexpressed in B. megaterium MS941 and demonstrated the applicability of this system for biotechnological purposes [65] (Table 1).…”

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes

“…The same approach has been used for microbial enzymes, especially to reduce the number of separate components in the electron transfer pathway (Bakkes et al, 2017) including fusing with: the reductase domain of P450 BM3 (Ugalde et al, 2018); mitochondrial adrenodoxin plus an E. coli ferredoxin reductase (Ringle et al, 2013); E. coli flavodoxin and flavodoxin reductase (Bakkes et al, 2015); the reductase domain of P450RhF (RhFRed) (Nodate et al, 2006;Li et al, 2007a;Sabbadin et al, 2010); and the reductase domain of CYP102D1 (Choi et al, 2014). The linker joining the two domains is often tweaked (Zuo et al, 2017) and in some studies, the redox partners interaction face has also been engineered (Sagadin et al, 2019). Fusions have also been explored with cofactor recycling systems e.g., phosphite dehydrogenase (Beyer et al, 2018).…”