Site-Directed Mutagenesis of Bile Salt Hydrolase (BSH) from Lactobacillus plantarum B14 Confirms the Importance of the V58 and Y65 Amino Acids for Activity and Substrate Specificity

Abstract

ABSTRACT The bile acids (BAs) de-conjugation is catalyzed by bile salt hydrolase (BSH) enzyme, that is an intestinal bacterial product and a member of the cholylglycine hydrolase (CGH) family. De-conjugated BAs alter BA-mediated signaling pathways such as glucose metabolism, energy homeostasis and lipid absorption and this makes the BSH clinically important. However, BSHs from different sources have a variable substrate preference to eight different bile salts. Although BSH is a well-studied enzyme, its molecular investigations based on BSH substrate recognition are not very well known. In this study, the relationship between substrate specificity of BSH from Lactobacillus plantarum B14 (LpBSH) and its loop II, the aliphatic-hydrophobic V58 and aromatic-hydrophobic Y65 residues in this loop was mutated and analyzed. While PCR-based site-directed mutagenesis was used to substitute V58 and Y65 amino acids for N58, F58, M58, C65, F65 and L65 amino acids, respectively, the BLR (DE3) strain of E. coli was used to express mutant recombinant LpBSHs (mrLpBSHs). Site-directed mutagenesis of LpBSH showed reduced activity of mrLpBSHs against six different BAs. Our results indicated that the V58 and mostly Y65 residues in loop II might be critical for the structural site that is involved in substrate specificity and catalysis. These findings suggested that V58 and Y65 residues of LpBSH might participate in substrate specificity and BSH substrate specificity may be dependent upon the collate group, rather than amino acid moieties. However, more mutagenesis-based investigation on other CGH family members are needed in order to understand the structure and substrate specificity relations of BSHs.