GST A1-1 Research Articles

Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species.

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the "H-site" in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.

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.

Structural and functional analysis of the inhibition of equine glutathione transferase A3-3 by organotin endocrine disrupting pollutants.

Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrine-disrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects.

Abstract 5238: Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer

Abstract Dendrogenin A (DDA) is a tumor suppressor metabolite identified in human tissues that arises from the conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA) by a yet unidentified enzyme. DDA is present in the normal breast but its levels were found drastically decreased in breast tumors, showing that a deregulation of DDA metabolism occurred during breast carcinogenesis. It was shown that DDA displayed chemopreventive and anticancer properties (de Medina et al, Nat Commun, 2013; Voisin et al, PNAS, 2017; Segala et al, Nat Commun, in press). In addition, DDA blocks the biosynthesis of a newly identified cholesterol tumor promoter named 6-oxo-cholestan-3β,5α-diol (OCDO) (Voisin et al, PNAS, 2017). DDA and OCDO arise from 5,6-EC. We showed the existence of a metabolic balance between these two 5,6-EC derivatives in normal breast and BC that controls or stimulates BC progression (Silvente-Poirot & Poirot, Science, 2014, Voisin et al, PNAS, 2017). We addressed here the question of the identification and characterization of the DDA synthase (DDAS) and we determined whether its expression could reflect DDA levels in patient breast tumor and normal tissue. We report that the recombinant human glutathione transferase A1-1 (GST A1-1) produced DDA from 5,6α-EC and histamine (HA). The chemical characterization of the DDA product was performed by chromatography and mass spectrometry fragmentation. DDAS activity was found to be a new and important activity of GST A1-1 in addition to known glutathione transferase and steroid isomerase activities. The measured Michaelis constants of GST A1-1 for its new substrates were: Km5,6α-EC=0.27±0.05 µM and KmHA=0.35±0.3 µM, and the maximum velocity for the transformation of each substrates Vm5,6α-EC=0.81±0.2 µmol.min-1.mg and VmHA=0.66±0.2 µmol.min-1.mg. Interestingly, we showed that OCDO and other ring-B oxysterols, as well as several natural substrates and product of the GST A1-1, were potent inhibitors of DDAS activity while xenobiotics substrates of GST, and side chain oxysterols were not. Patient BC samples (n=50) showed significant decreased DDA levels and lower GST A1-1 protein expression compared to normal matched tissues, indicating that the decreased production of DDA in tumors is due to decreased expression of its enzyme. The analyses of two human BC mRNA databases from the Barts Cancer Institute (London, UK) and the Curie Institute (Paris, France) showed that the expression of GST A1-1 was lost in ER(+) BC tumors compared to normal breast tissue. Interestingly, DDAS was selectively expressed in the cytoplasm of epithelial cells from lactating ducts and lobular terminal units. Since these cells are the origin of most BC, the loss of DDAS expression and DDA biosynthesis combine to OCDO production, which controls DDAS activity, may constitute a major oncogenic process leading to BC development in human. Citation Format: Marc Poirot, Emmanuel Noguer, Florence Dalenc, Regis Soules, Lisa Barrett, Arnaud Rives, Hye-Young Kim, Brigitta Sjödin, Camille Franchet, Pilippe Rochaix, Raphaelle Duprez-Paumier, Magali Lacroix-Triki, Thomas Filleron, Leonor Chaltiel, Louise Jones, Emanuala Gadaleta, Claude Chalala, Sergio Roman-Roman, Thierry Dubois, Ned A. Porter, Bengt Mannervik, Michel Record, Sandrine Silvente-Poirot. Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5238.

Characterization of equine GST A3-3 as a steroid isomerase

Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione and of Δ5-pregnene-3,20-dione to Δ4-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.

Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates

BackgroundOrganic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. MethodsIn the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. ResultsSignificantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. ConclusionsWe explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significanceThe facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.

Isomerization of Δ5-androstene-3,17-dione into Δ4-androstene-3,17-dione catalyzed by human glutathione transferase A3-3: a computational study identifies a dual role for glutathione.

Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ(5)-AD) and Δ(5)-pregnene-3,20-dione (Δ(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Δ(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Δ(5)-AD C4 atom to the Δ(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal · mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the O3 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leu111, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Δ(4)-AD crystal structure and is consistent with all available experimental data.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1

ObjectiveGlutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. MethodsWe tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. ResultsWe identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implicationsIn their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Structure-Based Redesign of GST A2-2 for Enhanced Catalytic Efficiency with Azathioprine

Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.

Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine

Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structure-activity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with ∼10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93% library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.

Abstract 3246: Synthesis and biochemical characterization of novel analogues of ezatiostat hydrochloride (TLK199, Telintra®)

Abstract Ezatiostat hydrochloride (TLK199, Telintra®) is the first glutathione S-transferase P1-1 (GST P1-1) inhibitor prodrug to demonstrate a clinically significant improvement in the cytopenias affecting patients with myelodysplastic syndrome (MDS), thereby validating this enzyme as a candidate target for pharmacologic intervention (Blood 2009; 113(26):6533-6540). Structurally, ezatiostat is a tripeptide glutathione analog diethyl ester that is metabolized in vivo to the diacid, which is a potent and selective inhibitor of GST P1-1, an enzyme overexpressed in many human hematologic cancers. This inhibition induces a dissociation of GST P1-1 from its complex with c-jun N-terminal kinase (JNK)/c-Jun and activates signaling pathways that lead to cell proliferation and differentiation of normal hematopoietic cells and apoptosis of malignant cells. To improve the inhibitory potency and selectivity for the target enzyme GST P1-1, a series of diacid analogs of ezatiostat bearing different substituents on the cysteinyl sulfur were synthesized and evaluated in several biological assays. Replacement of the benzyl group by a 2- or 4-biphenylmethyl or a 1-naphthylethyl substituent resulted in a 30- to 130-fold improvement in IC50 against GST P1-1 and 3- to 10-fold higher selectivity relative to the GST A1-1 and GST M1-1 isoforms. The most active analogs were esterified and tested in cell-based and in vivo assays. The esters induced growth arrest and cellular apoptosis in human leukemia cells (HL-60) with lower or similar CC50 values as ezatiostat (6-17 μM range). In an HL-60 differentiation assay measuring induction of cell surface CD11b expression, the compounds increased CD11b expression by approximately 2-fold. Similarly to ezatiostat, the compounds also increased the production of reactive oxygen species in HL-60 and 293T cells by 2- to 3-fold after a 2 hour exposure, as measured by the generation of a fluorescent signal from a redox-sensitive dye. Select ezatiostat analogs were evaluated in rodents for toxicity as well as for acceleration of recovery of neutrophil levels in a standard model of 5-FU chemotherapy-induced neutropenia. The compounds were well tolerated when administered at doses up to 200 mg/kg and, as ezatiostat, led to a significant >1 day accelerated recovery in the 5-FU-induced neutropenia model in rats compared to growth factor treatment. Substitution at the cysteinyl sulfur was established as the most critical position in the structure-activity relationship analysis and is consistent with the unique mechanism of action of these tripeptide inhibitors. Further investigation of the most potent and selective analogs in biochemical and cellular assays is underway. The availability of more selective and potent GST P1-1 inhibitors may help elucidate the role of GST P1-1 in the development and progression of MDS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2011-3246

Experimental conditions affecting functional comparison of highly active glutathione transferases

Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. The combination of time-dependent and enzyme concentration-dependent loss of activity and the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione transferases

Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.

Characterization of porcine Alpha-class glutathione transferase A1-1

An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1∗. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal

4-Hydroxy-2- trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it.

Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine

Azathioprine has been clinically used for decades in connection with organ transplantation, autoimmune disease, and treatment of cancer. Toxic side-reactions are common and have been linked to the liberation of excessively high concentrations of 6-mercaptopurine and corresponding toxic metabolites. An allelic variant of thiopurine methyltransferase with low activity is associated with elevated concentrations of 6-mercaptopurine. However, other genetic markers remain to be identified in order to fully account for adverse reactions and efficacy failure. In the present study, we studied the five known allelic variants of human glutathione transferase A2-2 (GST A2-2) (EC 2.5.1.18), abundantly expressed in liver and efficiently catalyzing the bioactivation of azathioprine to release 6-mercaptopurine. All five variants exhibited high activity with azathioprine, but allelic variant E of GST A2-2 displayed a 3–4-fold elevated catalytic efficiency compared to the other variants. High GST activity can lead to overproduction of 6-mercaptopurine, and the nature of the multiple forms of GSTs in a patient will obviously affect the metabolism of azathioprine. In addition to GST A2-2, the polymorphic GST M1-1 is also highly active with azathioprine. Considering our findings, it appears that the genotypic and phenotypic variations in the GST complement may influence the presentation of adverse reactions in patients treated with azathioprine. Clinical trials will be required to clarify the impact of the GST expression in comparison with the established biomarker thiopurine methyltransferase as predictors of adverse reactions.

Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Δ 5-androstene-3-17-dione into Δ 4-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.

Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with > 300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in k cat/ K m values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.

Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases

Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: 'specific catalytic capacity', 'substrate selectivity', and 'unit-scaled substrate selectivity'. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.