Sal is a proteobacterial bile acid aldolase that repurposes key thiolase catalytic residues for retroaldol cleavage of C5 steroid side chains

TL;DR

This paper describes a bacterial enzyme that can cleave steroid side chains, offering new insights into steroid metabolism and potential biocatalytic applications.

Contribution

The study identifies a novel C5 side chain steroid aldolase from Proteobacteria with unique catalytic residues and structural features.

Findings

CtSal uses Tyr302 and Cys304 as catalytic residues, differing from actinobacterial homologs.

CtShyDUF35 modulates CtSal's substrate specificity via a C3H1 zinc finger.

A homolog in Trypanosoma brucei suggests a repurposed thiolase-like aldolase function.

Abstract

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 C5 side chain of bile acid thioester steroids, whereas a previously characterized actinobacterial homolog from Thermonospora curvata (TcLtp2) targets the C3 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 (CtShyDUF35–CtSal) reveals a central CtSal dimer flanked by two CtShyDUF35 domains in an αββα arrangement. CtShyDUF35 has a unique Cys3His1 (C3H1) 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 C5 side chain steroid aldolase, revealing its unique catalytic features and expanding our understanding of steroid side chain catabolism in Proteobacteria.

Figures