Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from ATCC27952.

The characterization of cytochrome P450 CYP125A13 from was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min nmol, 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 P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of species.