Rolfe Nicolas, Schroeter Kurt L, Forrester Taylor Jb, Kimber Matthew S, Seah Stephen Yk
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
J Biol Chem. 2025 Jul 1;301(8):110439. doi: 10.1016/j.jbc.2025.110439.
Aldolases hold potential as biocatalysts for the synthesis of novel steroid pharmaceuticals. The steroid aldolase from Comamonas testosteroni (CtSal) forms a complex with C. testosteroni steroid hydratase (CtShy). CtSal cleaves the C side chain of bile acid thioester steroids, whereas a previously characterized actinobacterial homolog from Thermonospora curvata (TcLtp2) targets the C side chain. We identified Tyr302 and Cys304 as the catalytic residues in CtSal, different from the paired Tyr residues found in TcLtp2. The 1.95 Å structure of CtSal bound to the C-terminal domain of unknown function 35 (DUF35) of CtShy (CtShy-CtSal) reveals a central CtSal dimer flanked by two CtShy domains in an αββα arrangement. CtShy has a unique CysHis (CH) zinc finger that shapes the substrate-binding cleft of CtSal, preventing the binding of the flat cholesterol rings while accommodating the bent rings of bile acids. Phylogenetically, Sals and Ltp2s form separate clades and are distantly related to thiolases. Intriguingly, a Trypanosoma brucei homolog, annotated as a thiolase-like protein (TbSLP), shares the catalytic architecture of CtSal, suggesting an aldolase rather than a thiolase function. This study provides the first detailed characterization of a C side chain steroid aldolase, revealing its unique catalytic features and expanding our understanding of steroid side chain catabolism in Proteobacteria.
醛缩酶有潜力作为生物催化剂用于合成新型甾体药物。睾丸酮丛毛单胞菌（Comamonas testosteroni）的甾体醛缩酶（CtSal）与睾丸酮丛毛单胞菌甾体水合酶（CtShy）形成复合物。CtSal切割胆汁酸硫酯甾体的C侧链，而之前鉴定的弯曲嗜热放线菌（Thermonospora curvata）的肌动菌属同系物（TcLtp2）作用于C侧链。我们确定了CtSal中的催化残基为Tyr302和Cys304，这与TcLtp2中发现的成对Tyr残基不同。CtSal与CtShy的未知功能结构域35（DUF35）的C末端结构域结合（CtShy-CtSal）的1.95 Å结构显示，一个中央CtSal二聚体两侧是两个呈αββα排列的CtShy结构域。CtShy有一个独特的CysHis（CH）锌指，它塑造了CtSal的底物结合裂缝，阻止扁平胆固醇环的结合，同时容纳胆汁酸的弯曲环。从系统发育角度来看，醛缩酶和Ltp2s形成不同的进化枝，并且与硫解酶的关系较远。有趣的是，布鲁氏锥虫（Trypanosoma brucei）的一个同系物，注释为硫解酶样蛋白（TbSLP），与CtSal具有相同的催化结构，表明其具有醛缩酶而非硫解酶的功能。这项研究首次对C侧链甾体醛缩酶进行了详细表征，揭示了其独特的催化特征，并扩展了我们对变形菌中甾体侧链分解代谢的理解。
