紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。     

Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members

A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.     

Glutathione disulfide and S-nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

Mycobacterium tuberculosis resides within alveolar macrophages. These phagocytes produce reactive nitrogen and oxygen intermediates to combat the invading pathogens. The macrophage glutathione (GSH) pool reduces nitric oxide (NO) to S-nitrosoglutathione (GSNO). Both glutathione disulfide (GSSG) and GSNO possess mycobactericidal activities in vitro. In this study we demonstrate that M. tuberculosis thioredoxin system, comprises of thioredoxin reductase B2 and thioredoxin C reduces the oxidized form of the intracellular mycothiol (MSSM) and is able to efficiently reduce GSSG and GSNO in vitro. Our study suggests that the thioredoxin system provide a general reduction mechanism to cope with oxidative stress associated with the microbe’s metabolism as well as to detoxify xenobiotics produced by the host.     

Large‐scale synthesis of tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield

To biosynthesize the (3R,5S)‐CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)‐CDHH. The scale‐up synthesis of (3R,5S)‐CDHH was performed in 5000 L bioreactor with 400 g/L of (S)‐CHOH at 30°C, resulting in a space‐time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)‐CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612–620, 2017     

Genomic heterogeneity and instability in colorectal cancer: spectral karyotyping, glutathione transferase-Ml and ras

Genomic instability in cancer is frequently described as being either chromosomal instability or microsatellite instability, although when events within chromosomes are monitored, extensive intrachromosomal instability is also found. Spectral karyotyping was used to visualize how extensively genomic instability gives rise to intratumor genomic heterogeneity in sporadic colorectal carcinomas. Two factors were then examined which might relate to intrachromosomal instability in colorectal cancers: the presence of the glutathione transferase-Ml gene to detoxify potential carcinogens, and the presence of activated ras which has been associated with chromosomal instability when first expressed. Intrachromosomal genomic instability was previously determined by inter-(simple sequence repeat) PCR (inter-SSR PCR) and by fractional allelic loss rate for 348 markers. GSTM1 status was determined for each of 49 tumors through use of specific PCR, and 28 of the tumors showed the GSTM1 null genotype. A significant association was found between GSTMl-null status and elevated inter-(simple sequence repeat) PCR instability. In contrast, no association was found with fractional allelic loss rate. The first exons of the K-ras and H-ras oncogenes were sequenced in 72 colorectal cancers; 19 of the tumors had a mutation in codon 12 of the K-ras gene (24.5%), but no H-ras mutations were found. A weak correlation (p=0.10) was observed between mutant K-ras and inter-(simple sequence repeat) PCR genomic instability, and no association existed with fractional allelic loss rate.     

Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1

Background

The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.

Methods

Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.

Results

The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.

Conclusions

H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.

General significance

The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.     

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.     

Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1

The loop following helix α2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k_cat value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the α2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K_M^GSH 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the α2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the α2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k_cat can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.     

The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants

The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 °C to 45 °C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.     

A Novel Quasi-Species of Glutathione Transferase with High Activity towards Naturally Occurring Isothiocyanates Evolves from Promiscuous Low-Activity Variants

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.     

Transport of glutathione transferase-fold structured proteins into living cells

Glutathione transferases are a family of enzymes that are traditionally known to contribute to the phase II class of detoxification reactions. However, a novel property of the Schistosoma japonicum glutathione transferase (Sj.GST26) involves its translocation from the external medium into a variety of different cell types. Here we explore the efficiency and mechanism of cell entry for this class of protein. Using flow cytometry and confocal microscopy, we have examined the internalisation of Sj.GST26 into live cells under a variety of conditions designed to shed light on the mode of cellular uptake. Our results show that Sj.GST26 can effectively enter cells through an energy-dependent event involving endocytosis. More specifically, Sj.GST26 was found to colocalise with transferrin within the cell indicating that the endocytosis process involves clathrin-coated pits. A comprehensive study into the cellular internalisation of proteins from other classes within the GST structural superfamily has also been conducted. These experiments suggest that the ‘GST-fold’ structural motif influences cellular uptake, which presents a novel glimpse into an unknown aspect of GST function.     

Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential

Selenoprotein K (SelK) is a membrane protein involved in antioxidant defense, calcium regulation and the ER-associated protein degradation pathway. We found that SelK exhibits a peroxidase activity with a rate that is low but within the range of other peroxidases. Notably, SelK reduced hydrophobic substrates, such as phospholipid hydroperoxides, which damage membranes. Thus, SelK might be involved in membrane repair or related pathways. SelK was also found to contain a diselenide bond—the first intramolecular bond of that kind reported for a selenoprotein. The redox potential of SelK was −257 mV, significantly higher than that of diselenide bonds in small molecules or proteins. Consequently, SelK can be reduced by thioredoxin reductase. These finding are essential for understanding SelK activity and function.     

The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase

Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC–MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10–25 μM) showed 70–84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25 μM) demonstrated ?85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50 μM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC₅₀ of CAPE (15 μM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and l-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.