Site-directed mutagenesis of Bile Salt Hydrolase (BSH) from Lactobacillus plantarum B14 and substrate specificity analysis of Mutant BSH enzymes

Bile acid (BA) deconjugation is catalysed by the bile salt hydrolase (BSH) enzyme, which is a member of the cholylglycine hydrolase (CGH) family and produced by intestinal bacteria. The clinically significant BSH alters BA-mediated signalling pathways related to lipid absorption, energy homeostasis and glucose metabolism. Nevertheless, BSHs exhibit varied substrate preference to different bile salts across sources. Despite the considerable research on BSH, the investigations of its molecular mechanisms concerning BSH substrate recognition remain limited. This research aims to analyze the correlation between the substrate specificity of Lactobacillus plantarum B14's BSH enzyme (LpBSH) and two residues, V58 and Y65, in its loop II. These residues possess aliphatic-hydrophobic and aromatic-hydrophobic properties, respectively. PCR-based site-directed mutagenesis was utilised to substitute M58, F58, N58, F65, L65 and C65 amino acids for V58 and Y65, respectively. The mutant recombinant LpBSHs (mrLpBSHs) were expressed using the BLR (DE3) strain of E. coli and the activity of mrLpBSHs against six different BAs were detected. The study showed that the V58 and predominantly Y65 residues in loop II could play a vital role in the structural site that is responsible for substrate specificity and catalysis. The results suggest that the Y65 and V58 residues of LpBSH can be involved in substrate specificity. It was also observed that collate group, rather than amino acid moieties, may determine the substrate specificity of BSH. However, further mutagenesis-based research on other CGH family members is necessary to comprehend the structure and substrate specificity relationships of BSHs. Obtained results indicated that BSHs have developed the ability to identify BAs at the steroid nucleus of cholate as well as at the amino acid groups. As a result, it seems likely that the only requirements for binding might be that the cholate moieties and amino acids match and complement the substrate-binding pockets appropriately.