Characterization of a Promiscuous Glutamate Cysteine Ligase from Leptospira interrogans

N. SASONI; S.A. GUERRERO; A.A. IGLESIAS; D.G. ARIAS

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

SAIB

Glutathione (GSH) is the most abundant low molecular weight thiol in almost all eukaryotic cells as well as in proteo and cyanobacteria. It is synthesized enzymatically in two ATP Mg+2 dependent steps. First, glutamate cysteine ligase (GCL) establishes a peptide bond between cysteine and glutamate, forming -glutamylcysteine ( GC). Second, glutathione synthetase (GS) adds a glycine residue to the carboxy-terminus of GC, producing glutathione. Analysis of the genome sequences of Leptospira interrongans (the casusative agent of the leptospirosis) indicates the absence of the encoding-gene to GS. However, the presence of the gene that encodes GCL (LIC11812) leads us to believe that L. interrogans could has GC as redox buffer similar to Halobacteria and Lactic acid bacteria. Therefore, we cloned, expressed in recombinant form and characterized the functionality of GCL of L. interrogans. We showed that the enzyme is active in presence of their physiologic substrates (glutamate, cysteine and ATP), and also has the ability to use GTP, aspartic and serine, although in lower proportion. The enzyme has optimal activity at pH~7.5. Our results add value to the genome project information and contribute to the understanding of the redoxmetabolism present in this bacterium.