An improved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family

Abstract

Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Grampositive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria. Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms.

Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity

“…In contrast, Panigrahi et al (2014) have observed the presence of an indel mutation in the loop region of CGH from Gram-negative bacteria shortening the loop.…”

“…In conclusion, we have resolved the structure of a functional penicillin V acylase from the Gram-negative plant pathogen P. atrosepticum, which shows high specific activity for Pen V. Panigrahi et al (2014) have reported the classification of choloylglycine hydrolases from Gram-positive and Gram-negative bacteria into different clusters, based on a new binding site profile-based scoring system. The two groups show low sequence homology (<30%) and significant structural differences, including the loss of the tetramer assembly motif and inclusion of a periplasmic signal pre-peptide in Gram-negative CGHs.…”

“…Lambert et al (2008) and Panigrahi et al (2014) have classified CGHs from different bacteria and observed that the enzymes from Gram-negative bacteria cluster separately. In addition, penicillin acylases and their close homologues from http://dx.doi.org/10.1016/j.jsb.2015.12.008 1047-8477/Ó 2015 Elsevier Inc. All rights reserved.…”

Dissection of Catalytic Site in Crucial Gut Microbiome Enzyme: Bile Salt Hydrolase

“…Loop1, loop2 and loop3 allowed the entry of substrates into the active site of the enzyme and referred as 'Site A', whereas the site of glycine release is referred to as Site L as shown below ( Fig. 1b) (Panigrahi, Sule, Sharma, Ramasamy, & Suresh, 2014;Rossocha et al, 2005). With few exceptions, most of the gram positive and gram negative organisms possess the tetrameric form of BSH.…”

“…With few exceptions, most of the gram positive and gram negative organisms possess the tetrameric form of BSH. Distinct clustering of gram positive and gram negative bacterial BSH were observed owing to indel mutation (20)(21)(22) amino acids) in the tetramer loop region (Panigrahi et al, 2014). Theoretical prediction states that the presence of tetramer loop provides thermostability to gram positive BSHs as compared to gram-negative BSHs (Panigrahi et al, 2014).…”