Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A: A tRNA Synthetase Paralog from Leishmania donovani [Molecular Biophysics]

March 7th, 2014 by Manhas, R., Tripathi, P., Khan, S., Lakshmi, B. S., Lal, S. K., Gowri, V. S., Sharma, A., Madhubala, R.

Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia utilizing asparagine synthetase A (AsnA) and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation, using sequence based analysis, we had shown that Leishmania spp. possess asparagine tRNA synthetase paralog asparagine synthetase A (LdAsnA) that is ammonia dependent. Here, we report the cloning, expression, and kinetic analysis of AsnA from L. donovani. Interestingly, LdAsnA was both ammonia/glutamine dependent. To study the physiological role of AsnA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdAsnA showed a growth delay in mutants. However, chromosomal null mutants of LdAsnA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdAsnA is essential for survival of the Leishmania parasite. LdAsnA enzyme was recalcitrant towards crystallization so we instead crystallized and solved atomic structure of its close homolog from Trypanasoma brucei (TbAsnA) at 2.2 Å. A very significant conservation in active site residues is observed between TbAsnA and E. coli AsnA. It is evident that absence of LdAsnA homolog from humans and its essentiality for the parasites makes LdAsnA a novel drug target.
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