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Structural Characterization and Functional Validation of Aldose Reductase from the Resurrection Plant Xerophyta viscosa

Author:
Singh, Preeti, Sarin, Neera Bhalla
Source:
Molecular biotechnology 2014 v.56 no.11 pp. 971-978
ISSN:
1073-6085
Subject:
Escherichia coli, abiotic stress, affinity chromatography, aldehyde reductase, bioinformatics, circular dichroism spectroscopy, complementary DNA, crops, cytoplasm, genes, heavy metals, malondialdehyde, models, molecular weight, prediction, proteins, sodium chloride
Abstract:
Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K M (1.2 mM) and k cₐₜ (14.5 s⁻¹) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha–beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl₂), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.
Agid:
818840