Characterization of CYP125A13, the First Steroid C-27 Monooxygenase fromStreptomyces peucetiusATCC27952

Abstract

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...

The bacterial cytochrome P450 (CYP) CYP125 enzymes can competitively oxidise sitosterol in the presence of cholesterol

“…28 These CYP125 enzymes from Mmar and Mulc have not been extensively characterised, though evidence of binding of cholest-4-en-3-one and cholesterol has been established. 28 Other characterised CYP125 enzymes include two from Mycobacterium smegmatis , CYP125A3 and CYP125A4, 14,29 and CYP125A13 from Streptomyces peucetius , 30 all of which are capable of cholesterol side-chain oxidation. Recent genetic analysis reveals a generally high copy number of CYP125 enzymes within actinobacteria, suggesting an important role in the metabolic fitness of these organisms.…”