
Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome
Author(s) -
Muhammad Faheem,
Diogo Martins-de-Sa,
Julia Freitas Daltro Vidal,
Alice Cunha Morales Álvares,
J. Brandão-Neto,
Louise E. Bird,
Mark D. Tully,
Frank von Delft,
Betulia M. Souto,
Betânia Ferraz Quirino,
Sônia Maria de Freitas,
J.A.R.G. Barbosa
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep38031
Subject(s) - peptidoglycan , biochemistry , protease , cysteine protease , cell wall , small angle x ray scattering , biology , bacterial cell structure , protein domain , enzyme , metagenomics , homology modeling , cysteine , chemistry , bacteria , gene , genetics , physics , scattering , optics
A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5 M −1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.