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Cytochrome P450 monooxygenases (CYP, EC 1.14.14.1) belong to a large family of enzymes that catalyze the hydroxylation of various substrates. Here, we present the crystal structure of CYP105P2 isolated from Streptomyces peucetius ATCC27952 at a 2.1 Å resolution. The structure shows the presence of a pseudo-ligand molecule in the active site, which was co-purified fortuitously and is presumed to be a biphenyl derivative. Comparison with previously determined substrate-bound CYP structures showed that binding of the ligand produces large and distinctive conformational changes in α2-α3, α7-α9, and the C-terminal loop regions. This structural flexibility confirms our previous observation that CYP105P2 can accommodate a broad range of ligands. The structure complexed with a pseudo-ligand provides the first molecular view of CYP105P2-ligand interactions, and it indicates the involvement of hydrophobic residues (Pro82, Ala181, Met187, Leu189, Leu193, and Ile236) in the interactions between hydrophobic ligands and CYP105P2. These results provide useful insights into the structural changes involved in the recognition of different ligands by CYP105P2. [ABSTRACT FROM AUTHOR]

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 orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named CYP102A15 and CYP102A170, from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724, respectively, were cloned and expressed in E. coli . The genes are homologues of CYP102A1 from Bacillus megaterium . They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C 12 -C 20 , with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with K m 15.3 μM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 μM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.