Glutathione Transferases as Efficient Ketosteroid Isomerases

In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M−1s−1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3β-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s−1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.

Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

<p>The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.</p>

Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

<p>The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.</p>

Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

Allergological Importance of Invertebrate Glutathione Transferases in Tropical Environments

Glutathione-S transferases (GSTs) are part of a ubiquitous family of dimeric proteins that participate in detoxification reactions. It has been demonstrated that various GSTs induce allergic reactions in humans: those originating from house dust mites (HDM), cockroaches, and helminths being the best characterized. Evaluation of their allergenic activity suggests that they have a clinical impact. GST allergens belong to different classes: mu (Blo t 8, Der p 8, Der f 8, and Tyr p 8), sigma (Bla g 5 and Asc s 13), or delta (Per a 5). Also, IgE-binding molecules belonging to the pi-class have been discovered in helminths, but they are not officially recognized as allergens. In this review, we describe some aspects of the biology of GST, analyze their allergenic activity, and explore the structural aspects and clinical impact of their cross-reactivity.