Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506

Abstract

A glutathione S-transferase (GST) gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506 (namely PsGST), was cloned and expressed in Escherichia coli. The open reading frame of PsGST comprised 654 bp encoding a protein of 217 amino acids with a calculated molecular size of 24.3 kDa. The rPsGST possesses the conserved amino acid defining the binding sites of glutathione (G-site) and substrate binding pocket (H-site) in GST N_3 family. PsGST was expressed in E. coli and the recombinant PsGST (rPsGST) was purified by Ni-affinity chromatography with a high specific activity of 74.21 U/mg. The purified rPsGST showed maximum activity at 40 °C and exhibited 14.2% activity at 0 °C. It was completely inactivated at 50 °C for 40 min. These results indicated that rPsGST was a typical cold active GST with low thermostability. The enzyme was little affected by H2O2 and Triton X-100, and 50.2% of the remaining activity was detected in the presence of high salt concentrations (2M NaCl). The enzymatic Km values for CDNB and GSH was 0.22 mM and 1.01 mM, respectively. These specific enzyme properties may be related to the survival environment of Antarctic sea ice bacteria.

Ecology of cold environments: new insights of bacterial metabolic adaptation through an integrated genomic-phenomic approach

“…Furthermore, at 4 °C PhTAC125 is able to grow on 1-Chloro-2,4-Dinitrobenzene (CDNB) that is known to be bound by glutathione S-transferases (GSTs), versatile enzymes playing an important role as detoxifier of exogenous and xenobiotic compounds in many bacteria 42, 43 , including the Antarctic bacteria Pseudoalteromonas sp. ANT506, where GST was shown to be a typical cold active enzyme 44 . Remarkably, two GST-like genes (YibF) are present in the PhTAC125 genome but not in PspTB41.…”

“…Interestingly, despite most of the genes coding for the enzymes involved in the Glutathione metabolism are shared between PhTAC125 and PspTB41, except for trypanothione synthetase (3.5.1.78) and glutathionyl-spermidine synthase (6.3.1.8), the catabolism of several substrates such as Glutatione (GSH), L-Cysteine, Glycine, Putrescine, L-Ornithine, Spermidine and Spermine is strongly reduced in PspTB41 at 4 °C compared to PhTAC125 (Figure S1). This result indicates that PspTB41 strain is not able to efficiently use such substrates under cold conditions and it might be due to a reduced activity of the enzymes directly involved in the reactions 44 . Interestingly, together to GSH, glutathionylspermidine is likely to be a parallel defense strategy of PhTAC125 against oxidative stress and has been proposed to be more effective at preventing DNA damage induced by free radicals or oxidative species than GSH.…”

A glutathione peroxidase from Antarctic psychrotrophic bacteriumPseudoalteromonassp. ANT506: Cloning and heterologous expression of the gene and characterization of recombinant enzyme

“…8,9,10 Our earlier studies have revealed that glutathione S-transferase (GST) and SOD play the main roles in the protection mechanism against high salt concentration and low-temperature in the Antarctic bacterium Pseudoalteromonas sp. 11,12 Among other antioxidant enzymes, GPxs are the ubiquitous cytosolic ones, which can catalyze the reduction of H 2 O 2 and other organic peroxides. However, there are few reports about biochemical characterizations of the GPx protein.…”

Nanopartículas Sintetizadas por Bacterias Antárticas y sus Posibles Mecanismos de Síntesis

“…Algunas adaptaciones enzimáticas y no enzimáticas descritas en bacterias antárticas son glutatión s-transferasa (Shi et al, 2014), glutatión reductasa (Pugin et al, 2014), tioredoxina (Falasca et al, 2012) catalasas (Tribelli et al, 2012), super óxido dismutasas (Zheng et al, 2006), carotenoides (Dieser et al, 2010) y óxido reductasas (Madonna et al, 2006).…”