CONICET | Buscador de Institutos y Recursos Humanos

Soybean [Glycine max (L.) Merr] is known as one of the most relevant crops in our country because of the several usages such as raw material for export, oil production and cattle feeding. Citrate synthase enzyme (CS), which plays an important role in the glyoxylate cycle into the peroxisome and in the Krebs cycle into the mitochondria, catalyzes the condensation of oxaloacetate and acetyl-CoA. Several reports indicated that the overexpression of CS in roots increased the aluminum stress tolerance by organic acid internal accumulation and exudation. According to our previous evidence, CS also would facilitate the export of carbon derived from amino and other organic acids from soybean seed mitochondria to the plastids, site where fatty acid synthesis occurs. Thus, CS would play an important role in the generation of precursors for the biosynthesis of storage compounds during seed filling. As a promising target enzyme for crop improvement, this work studies the two mitochondrial CS isoforms found in soybean which differ from each other solely in three amino acidic residues. First, the enzymes were expressed in E. coli BL21 DE3 transformed with pET28a(+) carrying each synthetic gene and purified using Ni-NTA affinity columns. Each protein was detected at high levels in the soluble fraction of bacterial extracts and its molecular mass and quality after purification was as expected. We determined the optimum pH, substrate affinities, and the effect of several metabolites on enzyme activity using Ellman´s reagent coupled assay. Despite the high sequence similarity, our results indicated subtle differences that could indicate a differential contribution of both CS isoforms to the carbon flux during soybean seed maturation.

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