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1.
The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal alpha-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the beta1-alpha1 loop, the end of the alpha4 helix, and the C-terminal alpha9 helix. A few residue changes in GST A4-4 result in alpha9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix alpha9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the alpha9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.  相似文献   

2.
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.  相似文献   

3.
A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs.  相似文献   

4.
Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pK(a) of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pK(a) 7.1. In both cases the pK(a) was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pK(a) values > or = 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.  相似文献   

5.
6.
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 kcat/Km 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.  相似文献   

7.
A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the α-class isozymes of GST. It is characterized by kinetic properties intermediate between the α- and π-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P. et al. (1992) FEBS Lett., 313, 173–176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other α-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with Mr values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxidase suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.  相似文献   

8.
4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.  相似文献   

9.
Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K(R)) of 99 +/- 2 microM and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17beta-estradiol-3, 17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2.  相似文献   

10.
11.
Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.  相似文献   

12.
The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.  相似文献   

13.
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.  相似文献   

14.
目的 将人类Tudor-SN(tudor staphylococcal nuelease)蛋白SN(1~4)基因片段分别定向连入pGEX-4T-1质粒,使Tudor-SN蛋白sN各功能片段与(;"蛋白在大肠埃希菌BL21细胞内融合表达.方法 以重组质粒pSG5-Tudor-SN-flag为模板,PCR法扩增出目的 基因,利用EcoR Ⅰ和Sal Ⅰ双酶切法将目的 片段连接纠pGEX-4T-1载体卜,再将构建成功的GST-hTudor-SN-SN(1~4)重组质粒转化人大肠埃希菌BL-21内,IPTG诱导表达后再以考马斯亮蓝染色法检测GST融合蛋白的表达.结果 以单/双酶切和基因测序法鉴定构建的重组质粒均无误,考马斯亮监染色法观察到GST融合蛋白的正确表达.结论 重组原核GST.hTudor-SN-SN(1-4)质粒成功构建和表达.  相似文献   

15.
A bphK gene encoding glutathione S-transferase (GST) activity is located in the bph operon in Burkholderia sp. strain LB400 but its role in polychlorinated biphenyl (PCB) metabolism is unknown. This gene was over-expressed in Escherichia coli and an in vivo assay based on growth of E. coli containing GST activity was used to identify potential novel substrates for this enzyme. Using this assay, 4-chlorobenzoate (4-CBA) was identified as a substrate for the BphK enzyme. High pressure liquid chromatography analysis and chloride ion detection showed removal of 4-CBA and an equivalent increase of chloride in cell extracts when incubated with this enzyme. These results would indicate that this BphK enzyme has dechlorination activity in relation to 4-CBA and may have a role in protection of other Bph enzymes against certain chlorinated metabolites of PCB degradation.  相似文献   

16.
17.
The correlation between sequence diversity and enzymatic function was studied in a library of Theta class glutathione transferases (GSTs) obtained by stochastic recombination of fragments of cDNA encoding human GST T1-1 and rat GST T2-2. In all, 94 randomly picked clones were characterized with respect to sequence, expression level, and catalytic activity in the conjugation reactions between glutathione and six alternative electrophilic substrates. Out of these six different compounds, dichloromethane is a selective substrate for human GST T1-1, whereas 1-menaphthyl sulfate and 1-chloro-2,4-dinitrobenzene are substrates for rat GST T2-2. The other three substances serve as substrates for both enzymes. Through this broad characterization, we have identified enzyme variants that have acquired novel activity profiles that differ substantially from those of the original GSTs. In addition, the expression levels of many clones were improved in comparison to the parental enzyme. A library of mutants can thus display a distribution of properties from which highly divergent evolutionary pathways may emerge, resembling natural evolutionary processes. From the GST library, a clone was identified that, by the point mutation N49D in the rat GST T2-2 sequence, has a 1700% increased activity with 1-menaphthyl sulfate and a 60% decreased activity with 4-nitrophenethyl bromide. Through the N49D mutation, the ratio of these activities has thus been altered 40-fold. An extensive characterization of a population of stochastically mutated enzymes can accordingly be used to find variants with novel substrate-activity profiles and altered catalytic properties. Recursive recombination of selected sequences displaying optimized properties is a strategy for the engineering of proteins for medical and biochemical applications. Such sequential design is combinatorial protein chemistry based on remodeling of existing structural scaffolds and has similarities to evolutionary processes in nature.  相似文献   

18.
Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.  相似文献   

19.
Within the Alpha class of the mammalian glutathione transferases two variants of subunit interfaces exist. One is conserved among the A4 subunits, whereas the second one is found in all other members of the Alpha class. The ability of the two Alpha class subunit interfaces to adopt a functional heterodimeric structure has been investigated here.The heterodimer GST A1-4 was obtained by co-expression of the two human Alpha class subunits A1 and A4 in Escherichia coli. A histidine tail was added to the N terminus of the A1 subunit to facilitate the purification of the heterodimer. The heterodimer was formed in a small proportion implying that the efficiency of the hybridization between subunit A1 and A4 is less than the propensity for homodimer formation. The hybrid enzyme was stable at low temperatures, but the two subunits dissociated and reassociated into homodimers at 40 degrees C.Three different substrates were used for subunit-selective kinetic characterization of the GST A1-4 heterodimer: 1-chloro-2,4-dinitrobenzene, nonenal and Delta(5)-androstene-3,17-dione. Both subunit A1 and subunit A4 were active in GST A1-4, but the specific activities and k(cat) values were lower than the average values of the two parental isoenzymes. However, at high temperatures the subunits of the hybrid enzyme dissociated and formed homodimers, and the activities increased to expected values. Hence, the low activities of the individual subunits in the heterodimer were reversible. The non-additive kinetic properties of the subunits in the heterodimer therefore highlight the importance of fine-tuned subunit interactions for optimal catalytic efficiency of GST A1-1 and GST A4-4.  相似文献   

20.
Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.  相似文献   

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