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Journal of Bacteriology, November 2005, p. 7481-7491, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7481-7491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biochemical and Mutational Analysis of Glutamine Synthetase Type III from the Rumen Anaerobe Ruminococcus albus 8

Kensey R. Amaya,1 Svetlana A. Kocherginskaya,1 Roderick I. Mackie,1,2 and Isaac K. O. Cann1,2*

Department of Animal Sciences,1 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 618012

Received 23 April 2005/ Accepted 8 August 2005

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.

* Corresponding author. Mailing address: Department of Animal Sciences, 1207 W. Gregory Dr., University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-2090. Fax: (217) 333-8804. E-mail: icann{at}uiuc.edu.

Journal of Bacteriology, November 2005, p. 7481-7491, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7481-7491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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