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

Abstract

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H 2 O 2 ) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

An Unprecedented Number of Cytochrome P450s Are Involved in Secondary Metabolism in Salinispora Species

“…CYP154 members are involved in regio- and stereo-selective hydroxylation of different steroids [ 85 , 86 ]. CYP154 from Nocardia farcinica IFM10152 is a bifunctional enzyme with O -dealkylation and ortho -hydroxylation activities [ 87 ].…”

Crystal Structure and Biochemical Analysis of a Cytochrome P450 CYP101D5 from Sphingomonas echinoides

“…The kinetic parameters were calculated from the plot of the reaction rate versus the substrate concentration. Coupling efficiency was determined as the percentage of NADH utilized for product formation over the total consumption of NADH [ 65 ]. The kinetics analysis was performed using a non-linear regression analysis based on Michaelis–Menten kinetics using the OriginPro program (OriginLab Corporation, Northampton, MA, USA).…”

Recent developments in the enzymatic modifications of steroid scaffolds

“…59 Novel steroid hydroxylases were also characterized from bacteria, animals, and other kingdoms. 60 For example, CYP154C2 from Streptomyces avermitilis MA4680 catalyzed the C-2α hydroxylation of TES (10). 61 The novel CYP17A2 from Mastacembelus armatus (MA_CYP17A2), identified by genome mining, efficiently performed the C-17α hydroxylation of PG (12) (120.9 ± 4.2 mg L −1 in 7 days).…”