Department of Chemistry, University of Adelaide, SA 5005, Australia.
Department of Chemistry, University of Adelaide, SA 5005, Australia.
J Steroid Biochem Mol Biol. 2023 Dec;235:106406. doi: 10.1016/j.jsbmb.2023.106406. Epub 2023 Oct 2.
The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.
细菌细胞色素 P450（CYP）单加氧酶家族的成员 CYP125 催化固醇衍生物的氧化，包括胆固醇和植物固醇，作为它们分解代谢的初始激活步骤。然而，一些细菌物种含有多个编码 CYP125 酶的基因和其他催化胆固醇/胆甾-4-烯-3-酮羟化的 CYP 酶。一个重要的问题是，为什么这些细菌有不止一种具有重叠底物范围的酶，能够催化这些固醇的烷基链的末端氧化。为了进一步了解这些酶的作用，我们研究了来自海分枝杆菌的 CYP125A6 和 CYP125A7 与各种胆固醇类似物。这些在甾体的 A 和 B 环上有修饰，我们评估了这些酶与每种酶的底物结合和催化活性。CYP125A7 的结果与来自结核分枝杆菌的 CYP125A1 酶的报道结果相似。用 CYP125A6 观察到与胆固醇类似物的底物结合和催化活性的差异。例如，虽然胆甾醇硫酸盐可以与两种酶结合，但它仅被 CYP125A6 氧化，而不是被 CYP125A7 氧化。CYP125A6 生成了更多的代谢物，其中大部分是 C-3 和 C-7 取代的胆固醇类似物，如 7-酮胆固醇。然而，只有 CYP125A7 酶才能氧化 5α-胆甾烷-3β-醇。使用 AlphaFold 对 CYP125A6 和 CYP125A7 酶的胆甾-4-烯-3-酮和 7-酮胆固醇结合形式进行建模。结构模型突出了同一酶内固醇衍生物结合模式的差异。还观察到这些 CYP125 酶与其他细菌胆固醇氧化酶 CYP142A3 和 CYP124A1 之间的类固醇结合模式的显著变化。尽管如此，所有这些模型都预测了末端甲基羟化的选择性，这与实验数据一致。
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