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.

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase ( Ba CYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that Ba CYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of Ba CYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in Ba CYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant’s kcat/Kmvalues and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols.

During catalytic reactions, cytochrome P450 monooxygenase (P450) utilizes molecular oxygen to insert an oxygen atom into the substrate, which is usually an organic molecule. Simultaneously, the remaining oxygen atom is reduced to water [1]. The main feature of P450 enzymes is the hydroxylation of the carbon atoms of hydrophobic substrates. Similarly, the variety and flexibility of P450 enzymes make them nature's precious gift to a variety of chemical reactions [2]. They catalyze various types of chemical reactions like C-hydroxylation, heteroatom oxygenation, dealkylations, and epoxidations, making them appealing and versatile enzymes to produce various compounds with cosmetic and pharmaceutical value [3-5]. Most bacterial cytochrome P450s (CYPs) depend on electron transfer components (redox partners) comprised of NAD(P)H-dependent ferredoxin reductase (FDR) and ferredoxin (FDX), which sequentially deliver electrons from the cofactor to heme iron during catalysis [6]. Moreover, some CYPs such as peroxygenases from the CYP152 P450 family function efficiently, using hydrogen peroxide (H 2 O 2) without the need for redox partners [7, 8]. Examination of the genome sequence showed that Actinobacteria contained a large number of genes representing P450s, relative to other prokaryotes [9]. There are 20 P450 genes in Streptomyces peucetius alone. The exact function of most of these monooxygenases is still unknown. However, some are assumed to be involved in steroids and sterol (cholesterol) metabolism (Fig. 1). Cholesterol is an important compound, essential for cell membrane structure and fluidity, steroid hormone synthesis, and bile production in humans [10]. In the case of microorganisms, Mycobacterium tuberculosis (Mtb) contains many genes responsible for cholesterol metabolism. Mtb degrades cholesterol and utilizes it either for energy or as a biosynthetic precursor [11]. Mycobacterial Cyp125 (Rv3545c) is responsible for the hydroxylation of C27-steroids [12], and similarly CYP125 from Rhodococcus jostii RHA1 is a cholesterol hydroxylase orthologue to Mtb P450 (RV3545c) [13]. Cholesterol degradation in microbes involves two processes, steroid ring-opening and side-chain elimination [14]. Initially, it The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min −1 nmol −1 , and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This ori...

Cytochrome P450 enzymes (CYPs) are heme-containing enzymes that catalyze hydroxylation with a variety of biological molecules. Despite their diverse activity and substrates, the structures of CYPs are limited to a tertiary structure that is similar across all the enzymes. It has been presumed that CYPs overcome substrate selectivity with highly flexible loops and divergent sequences around the substrate entrance region. Here, we report the newly identified CYP101D5 from Sphingomonas echinoides. CYP101D5 catalyzes the hydroxylation of β-ionone and flavonoids, including naringenin and apigenin, and causes the dehydrogenation of α-ionone. A structural investigation and comparison with other CYP101 families indicated that spatial constraints at the substrate-recognition site originate from the B/C loop. Furthermore, charge distribution at the substrate binding site may be important for substrate selectivity and the preference for CYP101D5.