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Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes. [ABSTRACT FROM PUBLISHER]

Abstract: Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea. [Copyright &y& Elsevier]

Abstract: We identified a network of hydrogen bonds that is conserved in the structures of bacterial Beta class glutathione S-transferases (GSTs). It is formed by three residues: a serine, a histidine and a glutamate, together with a water molecule that links the serine with the histidine. This network connects the first helix of the N-terminal glutaredoxin-like domain with the last helix of the C-terminal GST-specific all helical domain. Here we show that substitution of Ochrobactrum anthropi GST His15 and Glu198 with alanine greatly compromises the catalytic efficiency of the enzyme, even though none of these residues takes part to the enzyme active site. Thermal and chemical denaturation experiments point to a role for this network in global structure stabilization. Furthermore, we show that OaGST structure looses compactness at alkanine pHs and that this behavior may be ascribed to partial disruption of the H-bond network, pointing to an important role in zippering the N-terminal and C-terminal domains of the protein. [Copyright &y& Elsevier]

Abstract: Glutathione transferases (GSTs) constitute a class of detoxifying enzymes involved in Phase II metabolism. Using GSH-affinity chromatografy followed by HPLC analysis, two GST isoforms were isolated from the Anguilla anguilla liver cytosol. The major GST belongs to the piscine-specific rho class and accounted for about 59% of total GST affinity eluted fraction, while the remaining 41% was represented by a Pi class GST. Both isoforms were cloned, heterologously expressed in Escherichia coli and their enzyme activities were characterized with respect to a broad spectrum of well-known GST substrates. Our data indicate that only a fraction of prototypical GST substrates are conjugated by these enzymes and that Pi class GST has higher specific activity than rho class GST against 1-chloro-2,4-dinitrobenzene (CDNB), ethracrynic acid, 4-nitroquinoline-1-oxide and p-nitrophenyl acetate while trans-2-nonenal is detoxified more efficiently by rho class GST. Analysis of the kinetics parameters of the conjugation against CDNB indicated that the utilization ratio K cat/K m is slightly higher for rho class GST with respect to pi class GSTs. Finally, to determine the potential for environmental inhibition of the GST isoforms, we examined the effect of the widely used herbicide atrazine as an inhibitor of catalytic activity. The inhibition studies revealed that atrazine was an effective inhibitor of GST–CDNB catalytic activities of both isoforms at micromolar concentrations, suggesting the sensitivity of these isoforms to pesticide inhibition at environmentally relevant concentrations. [Copyright &y& Elsevier]

Abstract: In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease. [Copyright &y& Elsevier]

To investigate structural relationship between amphibian and mammalian GSTs the complete amino acid sequence of the major form of glutathione transferase present in toad liver (Bufo bufo) was determined. The enzyme subunit is composed of 210 amino acid residues corresponding to a molecular mass of 24,178 Da. In comparison with the primary structure of amphibian bbGSTP1-1, toad liver GST showed 54% sequence identity. On the other hand, toad liver GST showed about 45–55% sequence identity when compared with other pi class GST and less then 25% identity with GST of other classes. Amino acid residues involved in the H site and in the key and lock structure of the toad enzyme are significantly different from those of bbGSTP1-1 and other mammalian pi class GST. On the basis of its structural and immunological properties the toad liver GST, indicated as bbGSTP2-2, could represent the prototype of a subset of the pi family. [Copyright &y& Elsevier]

Examines the properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase. Link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies; Reactivity of Cys10; Analysis of the redox properties of the Cys10.

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression. [Copyright &y& Elsevier]

Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin performed at 20°C and 30°C mainly generates two complementary peptides of 17 kDa and 6 kDa molecular mass with concomitant loss of catalytic capacity. Sequence analysis of these peptides showed that the peptide bond between Tyr47 and Gly48 was cleaved. The analysis of the recently resolved three-dimensional structure of GSTP1-1 [Reinemer, P., Dirr. H. W., Ladenstein, R., Huber, R., Lo Bello. M., Federici, G. & Parker, M. W. (1992) J. Mol. Biol. 227, 214–2261 suggests that the proteolytically cleaved bond results located in a portion of the polypeptide chain lining the G-site which has been demonstrated to be part of an exposed and flexible region of the N-terminal domain (structural elements αB′ and αB2 [Aceto. A., Caccuri, A. M., Sacchetta, P., Bucciarelli, T., Dragani, B., Rosato, N., Fedcrici, G. & Di Ilio, C. (1992) Biochem. J. 285, 241–245]. The fragments which are generated by proteolysis at 20°C, remain linked by noncovalent interaction in a complex (nicked GSTP1-1) which is dissociated by incubation at higher temperatures. As shown by circular dichroic analysis, although inactive, nicked GSTP1-1 retains an overall secondary structure closely resembling that of the parent enzyme. However, the fluorescence data of the nicked GSTP1-1 indicate that the Trp38, which is near the chymotrypsin-cleavable bond, becomes exposed in a more polar environment. This indicates that, in the nicked enzyme, the polypeptide portion containing the structural elements αB′ and αB2 has more freedom of fluctuation. The fact that this polypeptide chain portion contains two essential amino acid residues of the G-site (Trp38 and Lys42) might account for the loss of ability to bind glutathione by the nicked enzyme which is consequently catalytically inactive. Proteolysis performed at 30°C generated a homodimeric 17-kDa fragment. The structural analysis of this fragment suggests that the GSTP1-1 αC helix, which is located in the domain I and is thought to be involved in the inter-domain interaction, could exert a critical role in maintaining the native folding of domain II. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtained after cleavage by trypsin, Glu-C and Asp-N endoproteinases. The enzyme subunit of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17–26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes. [ABSTRACT FROM AUTHOR]