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Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

DOI: 10.1038/srep38031 DOI Help

Authors: Muhamamd Faheem (Universidade Catolica de Brasilia) , Diogo Martins-de-sa (Universidade de Brasília) , Julia F. D. Vidal (Universidade de Brasília) , Alice C. M. Álvares (Universidade de Brasília) , Jose Brandao-neto (Diamond Light Source) , Louise E. Bird (OPPF-UK, Research Complex at Harwell) , Mark D. Tully (Diamond Light Source) , Frank Von Delft (Diamond Light Source Ltd; Structural Genomics Consortium; University of Johannesburg) , Betulia M. Souto (Embrapa Agroenergia) , Betania F. Quirino (Universidade Católica de Brasília; Embrapa Agroenergia) , Sonia M. Freitas (Universidade de Brasília) , João Alexandre R. G. Barbosa (Universidade de Brasília)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Scientific Reports , VOL 6

State: Published (Approved)
Published: December 2016

Open Access Open Access

Abstract: 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 Kb of 1.8 × 105 M−1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg 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.

Journal Keywords: Membrane structure and assembly; Molecular modelling; Proteases; SAXS

Subject Areas: Biology and Bio-materials

Instruments: B21-High Throughput SAXS


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