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Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

ABSTRACT: Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the gamma-glutamyl transferase (gamma-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.

SUBMITTER: Amaya KR

PROVIDER: S-EPMC1272975 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

Amaya Kensey R KR Kocherginskaya Svetlana A SA Mackie Roderick I RI Cann Isaac K O IK

Journal of bacteriology 20051101 21

Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. ...[more]

PMID: 16237031

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Project description:BackgroundCellulose is the primary component of the plant cell wall and an important source of energy for the ruminant and microbial protein synthesis in the rumen. Cell wall content is digested by anaerobic fermentation activity mainly of bacteria belonging to species Fibrobacter succinogenes, Ruminicoccus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens. Bacteria belonging to the species Ruminococcus albus contain cellulosomes that enable it to adhere to and digest cellulose, and its genome encodes cellulases and hemicellulases. This study aimed to perform an in silico comparative characterization and functional analysis of cellulase from Ruminococcus albus to explore physicochemical properties and to estimate primary, secondary, and tertiary structure using various bio-computational tools. The protein sequences of cellulases belonging to 6 different Ruminococcus albus strains were retrieved using UniProt. In in silico composition of amino acids, basic physicochemical characteristics were analyzed using ProtParam and Protscale. Multiple sequence alignment of retrieved sequences was performed using Clustal Omega and the phylogenetic tree was constructed using Mega X software. Bioinformatics tools are used to better understand and determine the 3D structure of cellulase. The predicted model was refined by ModRefiner. Structure alignment between the best-predicted model and the template is applied to evaluate the similarity between structures.ResultsIn this study are demonstrated several physicochemical characteristics of the cellulase enzyme. The instability index values indicate that the proteins are highly stable. Proteins are dominated by random coils and alpha helixes. The aliphatic index was higher than 71 providing information that the proteins are highly thermostable. No transmembrane domain was found in the protein, and the enzyme is extracellular and moderately acidic. The best tertiary structure model of the enzyme was obtained by the use of Raptor X, which was refined by ModRefiner. Raptor X suggested the 6Q1I_A as one of the best homologous templates for the predicted 3D protein structure. Ramachandran plot analysis showed that 90.1% of amino acid residues are within the most favored regions.ConclusionsThis study provides for the first time insights about the physicochemical properties, structure, and function of cellulase, from Ruminococcus albus, that will help for detection and identification of such enzyme in vivo or in silico.

Dataset Information

Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

Publications

Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8.

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