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

Abstract

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H O , with optimum conversion surprisingly being achieved at ≈75 mm H O . More importantly, H O tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H O . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes

“…The regioselectivity of midazolam and caffeine metabolism was altered slightly but significantly when using organic peroxides compared to NADPH/CPRsupported reactions (Figure 2, Supplementary Tables 1 and 2). Similar observations have been made previously with various P450s and diverse substrates (Hrycay et al, 1975;Hanna et al, 2001;Yoshimoto et al, 2016;Dangi et al, 2018). This phenomenon is generally explained by a greater tendency of these OSs to undergo homolytic O-O bond scission as compared to H2O2, due to electron donating substituents (White and Coon, 1980;Barr et al, 1996;Nam et al, 2000).…”

“…More recent studies have aimed to optimize conditions for using peroxides to support P450 activities, but amongst human drug-metabolizing P450s, only CYP3A4 and CYP2D6 have been studied in any detail. No reports have examined iodosylbenzene derivatives for biocatalytic applications with drug-metabolizing P450s (Chefson et al, 2006;Kumar et al, 2006) although they have been explored with microbial forms (Dangi et al, 2018). Therefore, we set out to perform a more systematic analysis of the five major human xenobiotic-metabolizing P450s with several different commonly available OSs to determine the most appropriate OS to use for each form for both mechanistic studies and biocatalytic applications.…”

Characterization of high-H2O2-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

“…The well established P450 peroxygenase P450s SP (CYP152B1 from Sphingomonas paucimobilis), BS (CYP152A1 from Bacillus subtilis) and OleT (CYP152L1 from Jeotgalicoccus sp.) have been reported to replace the acid-alcohol amino acid pair, which is used for proton relay to iron-oxo species to facilitate the catalytic cycle (Munro et al, 2018 functioned in the presence of a high concentration of H 2 O 2 , as reported in our previous work (Dangi et al, 2018). The stability of heme also plays a crucial role in the efficient catalysis of the CYP.…”

Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 fromStreptomycesSpecies

“…Very few CYPs have shown activity in the presence of PIDA. CYP2B1, CYP3A4, CYP121, CYP101A1, CYP106A2, and CYP154C8 were mammalian and bacterial CYPs with catalytic activity in the presence of iodosobenzene [9,[49][50][51][52][53]. PIDA and iodosobenzene are single oxygen atom-containing surrogate oxidants and might share a similar monooxygenation mechanism [54].…”