Department of Chemistry, Marquette University, 1414W Clybourn Street, Milwaukee, WI, 53233, USA.
Departments of Biochemistry and Chemistry, University of Illinois, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL, 61801, USA.
Chemistry. 2020 Dec 15;26(70):16846-16852. doi: 10.1002/chem.202003181. Epub 2020 Nov 9.
Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C -C bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-O -O fragment, facilitating peroxo- attack on the C carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.
人细胞色素 P450 CYP17A1 首先催化 either pregnenolone (PREG) 或 progesterone (PROG) 在 C17 位的羟化，随后发生 C -C 键断裂，生成 dehydroepiandrosterone (DHEA) 或 androstenedione (AD)。在 T306A 突变体中，对羟化酶反应中必需的 Thr306 醇功能的取代，对裂合酶活性仅有很小的影响。在这项工作中，共振拉曼光谱学被用来提供关键的结构见解，证实了这个突变体，由于其无序的质子穿梭，无法生成必需的羟化酶途径中间体，导致羟化酶效率的丧失。重要的是，与易受影响的裂合酶底物 17-OH PREG 的相应光谱研究不仅揭示了最初捕获的过氧铁中间体经历了 17-OH 基团与 Fe-O-O 片段的近端氧的氢键相互作用，从而有利于过氧攻击 C 碳，而且还明确地显示了裂合酶反应的后续半缩酮中间体的存在。
