title: Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.
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 Kbof 1.8 × 105M-1for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rgof 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.
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.|Programa de Pós Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Faheem
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
authors.forename: Diogo
authors.initials: D
authors.lastname: Martins-de-Sa
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
authors.forename: Julia F D
authors.initials: JF
authors.lastname: Vidal
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
authors.forename: Alice C M
authors.initials: AC
authors.lastname: Álvares
authors.affiliation: Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11 0QX, England.
authors.forename: José
authors.initials: J
authors.lastname: Brandão-Neto
authors.affiliation: OPPF-UK, Research Complex at Harwell, Rutherford Appleton Laboratory, Oxford, OX11 0FA, United Kingdom.
authors.forename: Louise E
authors.initials: LE
authors.lastname: Bird
authors.affiliation: Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11 0QX, England.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Tully
authors.affiliation: Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11 0QX, England.|Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK.|Department of Biochemistry, University of Johannesburg, Auckland Park, 2006, South Africa.
authors.forename: Frank
authors.initials: F
authors.lastname: von Delft
authors.affiliation: Embrapa Agroenergia, Parque Estação Biológica - PqEB s/n°, Brasília, DF, 70770-901, Brazil.
authors.forename: Betulia M
authors.initials: BM
authors.lastname: Souto
authors.affiliation: Programa de Pós Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil.|Embrapa Agroenergia, Parque Estação Biológica - PqEB s/n°, Brasília, DF, 70770-901, Brazil.
authors.forename: Betania F
authors.initials: BF
authors.lastname: Quirino
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
authors.forename: Sonia M
authors.initials: SM
authors.lastname: Freitas
authors.affiliation: Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.|Programa de Pós Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil.
authors.forename: João Alexandre R G
authors.initials: JA
authors.lastname: Barbosa
mesh_terms: D000667:Ampicillin / Q000378:metabolism*; D000818:Animals; D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D057056:Cysteine Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006041:Goats / Q000382:microbiology*; D006868:Hydrolysis; D054892:Metagenome; D008958:Models, Molecular; D011485:Protein Binding; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D000667:Ampicillin; D057056:Cysteine Proteases