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.

中文翻译：

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CYP125A13 的表征，第一个类固醇 C-27 单加氧酶来自 Streptomyces peucetius ATCC27952。

使用胆固醇作为唯一底物对来自Streptomyces peucetius的细胞色素 P450 CYP125A13 进行表征。利用来自恶臭假单胞菌的恶臭还蛋白和恶臭还蛋白还原酶进行的体外酶促测定表明，CYP125A13 结合胆固醇并将其羟基化。计算得到的K _D值、催化转化率和Km值分别为56.92±11.28 μM, 1.95 nmol min ^-1 nmol ^-1, 和 11.3 ± 2.8 μM, 分别。气相色谱-质谱 (GC-MS) 分析显示胆固醇侧链的碳 27 被羟基化，将 CYP125A13 表征为类固醇 C27-羟化酶。同源性建模和对接结果还揭示了胆固醇与活性位点的结合，这是由疏水性氨基酸和 C27-甲基靠近血红素的位置促进的。这种取向有利于 C27-甲基的羟基化，支持体外分析。这是第一次报道链霉菌P450在 C-27 位羟基化胆固醇的案例。该研究还确定了 CYP125A13 的催化功能，并为进一步研究 CYP125A13 的分解代谢潜力提供了坚实的基础。链霉菌种。