Biochemical and Structural Characterization of Chi-Class Glutathione Transferases: A Snapshot on the Glutathione Transferase Encoded by sll0067 Gene in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Abstract

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in detoxification processes and/or in specialized metabolism. In the cyanobacterium Synechocsytis sp. PCC 6803, SynGSTC1, a chi-class GST (GSTC), is thought to participate in the detoxification process of methylglyoxal, a toxic by-product of cellular metabolism. A comparative genomic analysis showed that GSTCs were present in all orders of cyanobacteria with the exception of the basal order Gloeobacterales. These enzymes were also detected in some marine and freshwater noncyanobacterial bacteria, probably as a result of horizontal gene transfer events. GSTCs were shorter of about 30 residues compared to most cytosolic GSTs and had a well-conserved SRAS motif in the active site (10SRAS13 in SynGSTC1). The crystal structure of SynGSTC1 in complex with glutathione adopted the canonical GST fold with a very open active site because the α4 and α5 helices were exceptionally short. A transferred multipolar electron-density analysis allowed a fine description of the solved structure. Unexpectedly, Ser10 did not have an electrostatic influence on glutathione as usually observed in serinyl-GSTs. The S10A variant was only slightly less efficient than the wild-type and molecular dynamics simulations suggested that S10 was a stabilizer of the protein backbone rather than an anchor site for glutathione.

The Glutathione System: A Journey from Cyanobacteria to Higher Eukaryotes

“…The active site, located in a cleft between both domains, contains a GSH-binding site and a hydrophobic-substrate binding site. Based on their amino-acid sequence, GSTs were classified into various classes designated by a Greek letter [ 8 , 220 ]. GSTs having a sequence identity greater than 40% or lower than 25% belong to the same class or different classes, respectively [ 215 ].…”

“…That human orthologs of Sll1147 could rescue the stress tolerance of a Synechocystis PCC 6803 mutant lacking Sll1147 showed that the function of this MAPEG-type GST has been conserved during evolution from cyanobacteria to humans. The chi-class GST Sll0067 and its orthologs have been characterized biochemically from Thermosynechococcus elongatus BP-1, Synechococcus elongatus PCC 6301 and Synechocystis PCC 6803 [ 214 , 220 , 223 , 227 ]. Sll0067 purified from a recombinant E. coli strain as homo-dimer composed of about 20 kDa subunit appeared to have a good GSH-transferase activity with isothiocyanates (especially phenethyl isothiocyanate) and a low activity with the model substrate CDNB (1-chloro-2, 4-dinitrobenzene) [ 220 , 223 ].…”

“…The chi-class GST Sll0067 and its orthologs have been characterized biochemically from Thermosynechococcus elongatus BP-1, Synechococcus elongatus PCC 6301 and Synechocystis PCC 6803 [ 214 , 220 , 223 , 227 ]. Sll0067 purified from a recombinant E. coli strain as homo-dimer composed of about 20 kDa subunit appeared to have a good GSH-transferase activity with isothiocyanates (especially phenethyl isothiocyanate) and a low activity with the model substrate CDNB (1-chloro-2, 4-dinitrobenzene) [ 220 , 223 ]. Very interestingly, Sll0067 was found to play a crucial role in the detoxication of methylglyoxal ([ 214 ] and see below).…”

Versatility of Glutathione Transferase Proteins

“…The novel oxidizing conditions necessitated the emergence of protective agents such as glutathione, and it is a reasonable assumption that the evolution of GST was first established in cyanobacteria [22]. Mocchetti et al [23] have solved the crystal structure of the chi-class GST from the cyanobacterium Synechocystis, and furthermore, they have compared it with primary structures from other species. The chiclass proteins featured a well-conserved Ser-Arg-Ala-Ser amino acid motif in the active site of the N-terminal region, but a Ser residue appeared to be non-essential for catalytic activity, as indicated by mutagenesis of the Synechocystis GST.…”