3.1. Identification and characterization of three GS isoforms
Three different GS sequences (GS01, GS02 and GS03) have been identified through the sequencing and blastx searching. All the sequences contain a complete coding sequence (CDS) region and 5′ and 3′-UTRs. In this study we have attempted the characterization of the multiple GS cDNAs present. The characteristics details of the full-length cDNAs of GS01 (Accession No. JQ740737), GS02 (Accession No. JQ740738) and GS03 (Accession No. JX457351) are given in Table 2.
Analysis with the UTRscan tool revealed the presence of one Musashi Binding Element (MBE) in both GS01 and GS02 transcripts. But there was no MBE present in GS03 UTR. Conserved Domain Database search (CD-search) of amino acid sequences derived from the cDNA sequences exhibited two domains: a C terminal catalytic domain (Gln-synt_C) and an N terminal beta-Grasp domain (Gln-synt_N). Two sequence motifs were predicted by ScanProsite search: One is Glutamine synthetase signature 1 (aa position 62-79) and another Glutamine synthetase putative ATP-binding region signature (aa position 241-257). The motifs were conserved in all the three GS genes.
3.2. Structural analysis
The results of primary structure analysis of three GS (GS01, GS02 and GS03) sequences by ProtParam suggested a putative pI of 5.8, 6.7, 5.4 and MW of 41396.6, 41758.2, and 41588.7 kDa respectively. Motif Scan predicted presence of one N-glycosylation site (aa position 255-258), two cAMP and cGMP-dependent protein kinase phosphorylation sites (aa position 298-301 and 340-343), three Casein kinase II phosphorylation sites (aa position 53-56, 73-76, 193-196), and three Protein kinase C phosphorylation sites (aa position 257-259, 301-303, 322-324) in all the three GS proteins. In addition to that one more Casein kinase II phosphorylation (aa position 257-260) and Protein kinase C phosphorylation (aa position 322-324) sites in GS02 and GS03 proteins, and one Tyrosine kinase phosphorylation site (aa position 173-180) in GS01 and GS03 proteins were predicted. Presence of eight, and seven N-myristoylation sites were predicted for GS01, GS02/GS03 respectively. NetPhos 2.0 analysis projected 9/11/11 serine, 2/3/2 threonine and 5/7/7 tyrosine phosphorylation sites for GS01/GS02/GS03 proteins respectively. The homology modelling of the enzyme shows 12 identical subunits, arranged in two layers of 6. The secondary structure of GS consisted of 7 alpha helix and 15 beta strands. The binding residues, predicted by the RaptorX binding web server, and the corresponding ligands for the three different GS proteins are given in Table 3.
3.3. Phylogenetic analysis
The alignment of the multiple GS amino acid sequences with fishes, amphibians and mammalian proteins is presented in Fig. . The homologous active site residues for GS in C. batrachus were determined using the Salmonella typhimurium GS X-ray crystallography structure (Gill and Eisenberg, 2001). The pairwise alignment...