Functional characterization and bioinformatic analysis of glutamine synthetases of Ochrobactrum anthropi

Abstract

Glutamine synthetase (GS) is an essential enzyme required for the conversion of ammonium (NH4+) into glutamine. It is commonly classified into GSI, GSII, and GSIII based on molecular size, number of subunits, underlying regulation and enzyme structure. GSI is further subdivided into GSI-α and GSI-β. The latter form contains a conserved motif (NLYDLP) for the adenylation of a tyrosine residue near the active site and insertion of a specific 25-amino acid residue domain. Five ORFs were described to have GS activity in the genome of Ochrobactrum anthropi ATCC 49188. However, there are no studies regarding their functional significance and bioinformatic analysis. Therefore, with thorough bioinformatic analysis, I identified and classified these five putative distantly related GSs. Moreover, I modified the previously reported conserved motif (NLYDLP) for adenylation of tyrosine at the N-terminus of GSI-β to N/D-LYDLP. Using this modified motif as criterion as well as insertion of specific 25 amino acids, I identified the chromosome I GS (Oant_2087) of O. anthropi as GSI-β. Since those features were absent in the GS from pONAT01(Oant_4491) and the two GS of chromosome II (Oant_3936 and Oant_3881), they were identified as GSI-α. Chromosome II GS (Oant_4157) is GSII type. Further, my results from the bioinformatic analysis strongly indicate that GS on pONAT01 was acquired through horizontal gene transfer from either Ensifer adhaerens plasmid or Ensifer adhaerens chromosome 1. Interestingly, this transferred enzyme was found to be functional in O. anthropi as knocking it out from pONAT01 of O. anthropi resulted in 50% reduction in enzyme activity. All these findings will provide an insight the underlying mechanism of regulation for the five GSs present in O. anthropi and could serve as the basis for further investigation into the molecular functions of these five GSs, and the plasmid based one in particular.