Sal is a proteobacterial bile acid aldolase that repurposes key thiolase catalytic residues for retroaldol cleavage of C steroid side chains.

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.