Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy.
J Biol Chem. 2012 Jun 29;287(27):22626-34. doi: 10.1074/jbc.M112.372177. Epub 2012 May 17.
Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.
来自玫瑰色红球菌的甾体单加氧酶（STMO）利用 FAD 作为辅基和 NADPH 作为还原辅助因子，催化孕酮到孕酮醋酸盐的拜耳-维立格氧化反应。该酶与特征明确的拜耳-维立格单加氧酶（包括苯乙酮单加氧酶和环己酮单加氧酶）具有高度的序列相似性。STMO 的比较生物化学和结构分析对于理解拜耳-维立格单加氧酶的底物特异性特性特别有见地，这些特性正在成为生物催化应用中的有前途的工具，并作为前药激活的靶标。STMO 的天然、NADP(+)-结合和两种突变体形式的晶体结构揭示了这种微生物甾体降解酶的结构细节。与其他同类单加氧酶相比，NADP(+)的烟酰胺环的结合相对于黄素发生了移位。这一发现完全支持 NADP(H)在催化循环中采用各种位置来执行其在催化中的多种功能的观点。活性位点与苯乙酮单加氧酶非常相似。这一观察结果使我们发现 STMO 也能够作用于苯乙酮，这意味着它具有令人印象深刻的底物混杂性。对靶向底物结合位点表面残基的六个突变体的研究表明，孕酮和苯乙酮的酶促转化对相对剧烈的氨基酸变化基本不敏感，一些突变体甚至在孕酮上显示出增强的活性。这些特征可能反映了这样一个事实，即这些酶在不断进化以获得新的活性，这取决于生活环境中新型化合物的出现。
