Mutations of THR169 and PRO172 amino acids of BSH enzyme from lactobacillus plantarum and structural and functional analysis of the mutants

Bile salt hydrolase (BSH) (EC 3.5.1.24) has a very important role in host metabolism. Glycine and taurine in bile salts can be liberated by BSH which is called deconjugation. This property of BSH exert health benefits to the host such as reduction of blood cholesterol. Therefore BSH active probiotic strains are used in the treatment of hypercholesterolemia related diseases. Moreover, using BSH active strains as an alternative to antibiotic growth promoter (AGP) is under research. However, deconjugation of bile salts may have side effects to the host. Studies showed the relationship between BSH and a couple of diseases; colorectal cancer, cholesterol gallstone and bowel diseases. For this reason, understanding the mechanism of BSH enzyme is crucial. Even though BSH effects on human are well studied, there are not enough information about its mechanism of catalytic activity, substrate preferences and folding. In this study, two conserved amino acids, Thr-169 and Pro-172 from Lactobacillus plantarum B14 strain were substituted for valine and histidine respectively in pCON1 construct by site-directed mutagenesis then mutant bsh genes were inserted into the pET22b expression vector, and expressed in Escherichia coli BRL(DE3) strain. Mutant BSH enzyme activities were determined by ninhydrin assay with six different human bile salts, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid. The results showed that T169V mutation decreased the activity of BSH. Distinctly; inactive enzyme was obtained from P172H mutant. Meanwhile, stability and folding of the BSH proteins were not affected by these mutations. Our finding demonstrated that these two amino acids might be responsible for catalytic activity of BSH enzyme but not for stability of it.