Hernández-Fernández Gabriel, Ibero Juan, García José L, Galán Beatriz
Department of Biotechnology, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Community of Madrid, Spain.
Department of Biochemistry and Molecular Biology, Chemical Sciences Faculty, Complutense University of Madrid, Madrid, Community of Madrid, Spain.
mBio. 2025 Jul 9;16(7):e0107725. doi: 10.1128/mbio.01077-25. Epub 2025 May 30.
Progesterone (PROG) is one of the most ubiquitous sexual hormones found as a pollutant in soil and water systems. Despite the fact that PROG can be degraded by various bacterial species, the pathways leading to its complete oxic mineralization remain unknown. In this study, we investigated bacterial progesterone catabolism using the steroid-degrading bacterium as a model, in which we demonstrated its capacity to degrade progestogens. The transcriptomic analyses in the presence of PROG showed the overexpression of and genes, coding a Baeyer-Villiger monooxygenase (BVMO) and a luciferase-like monooxygenase (LLM), respectively. Both genes are located next to two regulatory proteins forming a small gene cluster (named ) that can be found in other steroid-degrading bacteria. Mutagenic analyses and gene complementation allowed ascertaining that and genes are involved in PROG degradation. To assess their enzymatic activities, both proteins were overexpressed in showing that they catalyze a Baeyer-Villiger monooxygenation of PROG, resulting in the production of testosterone acetate. They are also active on 1,2-dehydroprogesterone, an intermediate in PROG degradation, converting it into boldenone acetate. BVMO and LLM enzymes are functionally redundant, as each can replace the other in metabolizing PROG. The presence of two functionally redundant BVMO and LLM enzymes in can be explained by their distinct substrate preferences. Our results settle for the first time the genetic and biochemical basis for explaining how PROG is recognized and channeled into the 9,10-seco degradation pathway in a PROG-degrading bacterium.IMPORTANCEThis study investigates for the first time the key steps in bacterial progesterone (PROG) degradation, revealing new insights into the process. The main stages of PROG degradation were examined in the bacterium . The conducted transcriptomic analysis allows identifying the progesterone degradation cluster which is also present in other related bacteria. We demonstrated that Baeyer-Villiger monooxygenase and luciferase-like monooxygenase encoded within the cluster catalyze the PROG Baeyer-Villiger monooxygenation, producing testosterone acetate. The activity redundancy can be explained by the difference in substrate specificity of each enzyme.
孕酮（PROG）是在土壤和水系统中作为污染物发现的最普遍存在的性激素之一。尽管PROG可被多种细菌降解，但其完全有氧矿化的途径仍不清楚。在本研究中，我们以类固醇降解细菌为模型研究细菌孕酮分解代谢，证明了其降解孕激素的能力。在PROG存在下的转录组分析显示，分别编码拜耳-维利格单加氧酶（BVMO）和荧光素酶样单加氧酶（LLM）的基因和基因过表达。这两个基因位于形成一个小基因簇（命名为）的两个调节蛋白旁边，该基因簇也存在于其他类固醇降解细菌中。诱变分析和基因互补确定基因和基因参与PROG降解。为了评估它们的酶活性，两种蛋白质都在中过表达，表明它们催化PROG的拜耳-维利格单加氧反应，产生醋酸睾酮。它们对PROG降解的中间体1,2-脱氢孕酮也有活性，将其转化为醋酸勃地龙。BVMO和LLM酶在功能上是冗余的，因为它们在代谢PROG时可以相互替代。中两种功能冗余的BVMO和LLM酶的存在可以通过它们不同的底物偏好来解释。我们的结果首次确定了解释PROG在PROG降解细菌中如何被识别并导入9,10-开环降解途径的遗传和生化基础。重要性本研究首次调查了细菌孕酮（PROG）降解的关键步骤，揭示了该过程的新见解。在细菌中检查了PROG降解的主要阶段。进行的转录组分析允许识别在其他相关细菌中也存在的孕酮降解簇。我们证明，簇内编码的拜耳-维利格单加氧酶和荧光素酶样单加氧酶催化PROG的拜耳-维利格单加氧反应，产生醋酸睾酮。活性冗余可以通过每种酶底物特异性的差异来解释。
