Environmental Molecular Biology Laboratory, RIKEN, Saitama, Japan
Condensed Molecular Materials Laboratory, RIKEN, Saitama, Japan.
Appl Environ Microbiol. 2019 Oct 1;85(20). doi: 10.1128/AEM.01204-19. Print 2019 Oct 15.
TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3)-3-hydroxylacyl CoA-dehydrogenase, encoded by (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7β,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between and , encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of to , some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters. Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in TA441 as a model of steroid-degrading bacteria. is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire β-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.
TA441 通过 A 环的芳香化作用降解类固醇，然后降解 9,17-二酮-1,2,3,4,10,19-六降胆烷-5-酸，主要通过β-氧化。在这项研究中，我们揭示了 7β,9α-二羟基-17-氧代-1,2,3,4,10,19-六降胆烷-5-酸辅酶 A（CoA）酯由（3）-3-羟基酰基辅酶 A 脱氢酶（由 ORF27 编码）脱氢，然后得到的 9α-羟基-7,17-二酮-1,2,3,4,10,19-六降胆烷-5-酸-CoA 酯由 3-酮酰基辅酶 A 转移酶（由 ORF23 编码）转化。有了这些结果，阐明了 9,17-二酮-1,2,3,4,10,19-六降胆烷-5-酸侧链 C-8 上的整个β-氧化循环；ScdC1C2 使 9-羟基-17-氧代-1,2,3,4,10,19-六降胆烷-5-酸-CoA 酯在 C-6 脱氢，然后由 ScdD 水合。7β,9α-二羟基-17-氧代-1,2,3,4,10,19-六降胆烷-5-酸-CoA 酯然后由 ScdE 脱氢，转化为 9α-羟基-17-氧代-1,2,3,4,5,6,10,19-辛降胆烷-7-酸-CoA 酯和乙酰辅酶 A 由 ScdF 完成。ScdF 是 H37Rv 中 FadA6 的同源物，据报道它是参与 C 环裂解的 3-酮酰基辅酶 A 转移酶。我们还获得了表明 ScdF 也参与 C 环裂解的结果，但需要进一步研究证实。位于和之间的 ORF25 和 ORF26 编码属于酰胺酶超家族的酶。破坏 ORF25 或 ORF26 都不会影响类固醇降解。在具有与至相似基因簇的细菌中，有些既有 ORF25-和 ORF26-样蛋白，也有 ORF26-样蛋白，而有些则没有 ORF25-或 ORF26-样蛋白。ORF25 和 ORF26 对于类固醇降解不是至关重要的，但它们可能为阐明细菌类固醇降解簇的进化提供线索。细菌类固醇降解的研究始于 50 多年前，主要是为了获得类固醇药物的材料。类固醇降解细菌在全球范围内分布，细菌类固醇降解在环境中的作用以及与人类健康的关系引起了人们的关注。已经提出了四种基本甾体环的需氧降解；然而，要了解完整的降解途径，还有很多需要揭示。本研究旨在揭示 TA441 作为类固醇降解细菌的模型的整个类固醇降解过程。是研究最多的代表性类固醇降解细菌之一，适合探索降解途径，因为可以通过基因破坏来确定降解相关基因的参与。在这里，我们阐明了裂解 B 环的整个β-氧化循环。这个循环对于随后的 C 和 D 环裂解是必不可少的。
