Purification, characterization, and expression of multiple glutamine synthetases from Prevotella ruminicola 23.
The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa. Optimal conditions for γ-glutamyl transferase activity were found to be 35°C at pH 5.6 with 0.25 mM Mn(2+) ions (GSI) or 37°C at pH 6.0 (GSIII-1 and GSIII-2) with 0.50 to 1.00 mM Mn(2+) ions. GSIII biosynthetic activity was found to be optimal at 50 to 60°C and pH 6.8 to 7.0 with 10 mM Mn(2+) ions, while GSI displayed no GS biosynthetic activity. Kinetic analysis revealed K(m) values for glutamate and ammonium as well as for hydrolysis of ATP to be 8.58, 0.48, and 1.91 mM, respectively, for GSIII-1 and 1.72, 0.43, and 2.65 mM, respectively, for GSIII-2. A quantitative reverse transcriptase PCR assay (qRT-PCR) revealed GSIII-2 to be significantly induced by high concentrations of ammonia, and this corresponded with increases in measured GS activity. Collectively, these results show that both GSIII enzymes in P. ruminicola 23 are functional and indicate that GSIII-2, flanked by GOGAT (gltB and gltD genes), plays an important role in the acquisition and metabolism of ammonia, particularly under nonlimiting ammonia growth conditions.
Department of Animal Sciences, University of Illinois, Urbana, Illinois, USA.
Jong Nam
JN
Kim
Isaac K O
IK
Cann
Roderick I
RI
Mackie
D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005974:Glutamate-Ammonia Ligase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010802:Phylogeny; D045243:Prevotella ruminicola / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D005974:Glutamate-Ammonia Ligase