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• W2018736093 abstract "The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with kcat/Km values of 5 × 105 m-1 s-1 and 7 × 105 m-1 s-1, respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids. The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with kcat/Km values of 5 × 105 m-1 s-1 and 7 × 105 m-1 s-1, respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids. The glyoxalase system is a ubiquitous pathway for the detoxication of highly reactive ketoaldehydes. Glyoxalase I (EC 4.4.1.5., lactoylglutathione methylglyoxal lyase) and glyoxalase II (EC 3.1.2.6., hydroxyacylglutathione hydrolase) catalyze the dismutation of 2-ketoaldehydes into the corresponding 2-hydroxy acids using glutathione as cofactor (1Vander Jagt D.L. Dolphin D. Avramovic O. Poulson R. Coenzymes and Cofactors. IIIA. John Wiley & Sons, New York1989: 597-641Google Scholar, 2Thornalley P.J. Chem. Biol. Interact. 1998; 111–112: 137-151Crossref PubMed Scopus (283) Google Scholar). The main physiological function of the glyoxalase system is probably the detoxication of methylglyoxal, a mutagenic and cytotoxic compound that is mainly formed as a by-product of glycolysis. In addition, methylglyoxal is produced by the catabolism of amino acids via aminoacetone or hydroxyacetone (3Phillips S.A. Thornalley P.J. Eur. J. Biochem. 1993; 212: 101-105Crossref PubMed Scopus (473) Google Scholar). Methylglyoxal can react with the nucleophilic centers of DNA, RNA, and proteins. The ketoaldehyde reacts with the side chains of arginine, lysine, and cysteine and with the base guanine and to a lesser extent with adenine and cytosine. Glyoxalase I (GLX I) 1The abbreviations used are: GLX I, glyoxalase I; GLX II, glyoxalase II; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. converts the hemithioacetal, which is formed spontaneously from methylglyoxal and glutathione, into S-lactoylglutathione. The thioester is subsequently hydrolyzed by glyoxalase II (GLX II) yielding d-lactate and regenerating glutathione. Trypanosomatids are the causative agents of severe tropical diseases such as African sleeping sickness (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense), Nagana cattle disease (T. brucei brucei and Trypanosoma congolense), Chagas' disease (Trypanosoma cruzi), and the three manifestations of leishmaniasis (Leishmania donovani, Leishmania major, and Leishmania mexicana). All these parasitic protozoa have in common that the nearly ubiquitous glutathione/glutathione reductase system is replaced by trypanothione (N1,N8-bis(glutathionyl)spermidine) and the flavoenzyme trypanothione reductase (4Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (691) Google Scholar, 5Müller S. Liebau E. Walter R.D. Krauth-Siegel R.L. Trends Parasitol. 2003; 19: 320-328Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). A linkage between glutathione and spermidine metabolism was first discovered in Escherichia coli (6Tabor H. Tabor C.W. J. Biol. Chem. 1975; 250: 2648-2654Abstract Full Text PDF PubMed Google Scholar). During stationary phase, all of the cellular spermidine and a large part of glutathione occur as mono(glutathionyl)spermidine whereby the bacterium does not form trypanothione. The trypanothione metabolism is essential for the parasite (7Krieger S. Schwarz W. Ariyanayagam M.R. Fairlamb A.H. Krauth-Siegel R.L. Clayton C. Mol. Microbiol. 2000; 35: 542-552Crossref PubMed Scopus (314) Google Scholar) and is involved in the detoxication of hydroperoxides, the synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase, as well as the homeostasis of ascorbate (5Müller S. Liebau E. Walter R.D. Krauth-Siegel R.L. Trends Parasitol. 2003; 19: 320-328Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The pathogenic form of T. brucei multiplying in the blood of the mammalian host depends on glycolysis as the sole energy source and has a very high glucose turnover, which is about 200–300-fold higher than in erythrocytes (8Fairlamb A.H. Opperdoes F.R. Morgan M.J. Carbohydrate Metabolism in Cultured Cells. Plenum Publishing Corp., New York1986: 183-224Crossref Google Scholar). In E. coli and human red blood cells, the glycolytic rate has been shown to quantitatively correlate with the formation of methylglyoxal (2Thornalley P.J. Chem. Biol. Interact. 1998; 111–112: 137-151Crossref PubMed Scopus (283) Google Scholar, 3Phillips S.A. Thornalley P.J. Eur. J. Biochem. 1993; 212: 101-105Crossref PubMed Scopus (473) Google Scholar). These findings together with the fact that trypanothione instead of glutathione is the main low molecular mass thiol prompted us to characterize the glyoxalase system in African trypanosomes. The T. brucei genome contains two probable glyoxalase II sequences. Here we report on the cloning and overexpression of a glyoxalase II gene encoded on chromosome VI. The recombinant T. brucei protein slowly hydrolyzes lactoylglutathione, the substrate of classical glyoxalases II, but strongly prefers trypanothione thioesters. We provide strong evidence that glutathione is replaced by trypanothione also in the glyoxalase system of trypanosomatid parasites. S-Lactoylglutathione, methylglyoxal, yeast glyoxalase I (400–800 units/mg), bovine liver glyoxalase II (10 units/mg), and Leuconostoc mesenteroidesd-lactate dehydrogenase were purchased from Sigma. Hog muscle l-lactate dehydrogenase was from Roche. T. cruzi trypanothione reductase was prepared as described (9Sullivan F.X. Walsh C.T. Mol. Biochem. Parasitol. 1991; 44: 145-147Crossref PubMed Scopus (102) Google Scholar). Trypanothione disulfide was obtained from Bachem. Restriction enzymes and PfuTurbo DNA polymerase were from MBI Fermentas. Primer synthesis and DNA sequencing were performed by MWG Biotech. All other chemicals were commercially available reagents of the highest quality. T. brucei His6-GLX II—The coding region of the glxII gene was amplified from T. brucei genomic DNA (strain TREU 927/4 (10Melville S.E. Leech V. Gerrard C.S. Tait A. Blackwell J.M. Mol. Biochem. Parasitol. 1998; 94: 155-173Crossref PubMed Scopus (94) Google Scholar)) by PCR using sequence-specific primers derived from the data base. The 5′ primer (5′-gcgcggatccgaagttgtagtgaagagcatcgg-3′) contained a BamHI site (underlined) and gaa encoding glutamate 2 of the protein. The 3′ primer (5′-taggtaccacaccatagttcgcg-3′) was placed in the 3′-untranslated region directly after the stop codon. The gene was amplified from genomic DNA by PCR (95 °C for 2 min; 95 °C for 30 s; 55 °C for 30 s; 72 °C for 2 min; 30 cycles; 72 °C for 10 min; Pfu). The PCR product was digested with BamHI and cloned into the pQE-30 vector (Qiagen), digested with BamHI and SmaI. E. coli NovaBlue cells (Novagen) were transformed with the pQE-30/his-glxII plasmid. The plasmid was isolated using the NucleoBond® plasmid purification kit (Macherey-Nagel) and the insert was completely sequenced in both directions. For overexpression of T. brucei His6-GLX II, a 1-liter culture of recombinant NovaBlue cells was incubated at 37 °C in LB-medium containing 100 μg/ml carbenicillin. At an A600 of about 0.5, expression was induced by adding 200 μm isopropyl-β-d-thiogalactopyranoside and the cells were allowed to grow overnight at 15 °C. T. brucei GLX II—For overexpression of tag-free T. brucei glyoxalase II, the 5′ primer (5′-cgcgccatggaagttgtagtgaagagcatc-3′) contained an NcoI site (underlined) and the start ATG (italic). The 3′ primer (5′-cgcaggatccttacggacacgtattatagaggaag-3′) contained the stop codon (tta) and a BamHI site (underlined). The gene was amplified from genomic DNA (95 °C for 2 min; 95 °C for 30 s; 64 °C for 30 s; 72 °C for 2 min; 30 cycles; 72 °C for 10 min; Pfu) and the PCR product was cloned using the NcoI and BamHI restriction sites into the pQE-60 vector, resulting in the pQE-60/glxII plasmid. The plasmid was isolated and sequenced. NovaBlue cells were transformed with the plasmid and the gene was overexpressed as described above. The fusion protein, carrying a 14-residue long N-terminal extension with six histidine residues, was purified at 4 °C by chromatography on a TALON® metal affinity resin column (Clontech). Cells from a 1-liter bacterial culture were harvested by centrifugation, resuspended in 50 mm sodium phosphate, 300 mm NaCl, 1 mm imidazol, pH 7.0, 150 nm pepstatin, 4 nm cystatin, and 100 μm phenylmethylsulfonyl fluoride and lysed by sonification, and the cell debris was removed by centrifugation at 33,000 × g. The supernatant was applied onto a 5-ml resin pre-equilibrated in 50 mm sodium phosphate, 300 mm NaCl, 1 mm imidazol, pH 7.0. After washing the column with the equilibration buffer, followed by 50 mm sodium phosphate, 300 mm NaCl, 5 mm imidazol, pH 7.0, the protein was eluted with 250 mm imidazol in 50 mm sodium phosphate, 300 mm NaCl, pH 7.0. The recombinant T. brucei His6-glyoxalase II was ≥95% pure as judged by SDS-polyacrylamide gel electrophoresis. The protein concentration was determined using the bicinchoninic acid kit (BCA, Pierce). The recombinant protein without tag was purified on a Q-Sepharose fast flow cation exchanger (Amersham Biosciences). The column (10 ml) was equilibrated at 4 °C in 100 ml of 10 mm MOPS, pH 7.5. The recombinant NovaBlue cells from a 1-liter culture were harvested by centrifugation, the cell pellet was suspended in 5 mm MOPS, pH 7.5, 150 nm pepstatin, 4 nm cystatin, and 100 μm phenylmethylsulfonyl fluoride and lysed as described above. The supernatant was diluted with H2O to a conductivity lower than that of the equilibration buffer and applied onto the column. After washing the Q-Sepharose with 120 ml of equilibration buffer, the protein was eluted with 90 ml of 20 mm NaCl in 10 mm MOPS, pH 7.5, collecting 5-ml fractions. The purity of the protein was ≥95% as judged by SDS-polyacrylamide gel electrophoresis. For homogeneous His6-GLX II and tag-free GLX II a protein concentration of 0.58 mg/ml corresponds to a ΔA280 of 1. For long-term storage at -20 °C, 50% glycerol was added to the protein preparations. The subunit composition of T. brucei glyoxalase II was determined by fast protein liquid chromatography on a High Load 26/60 Superdex 75 column (Amersham Biosciences). Purified, tag-free protein (3.6 mg) was loaded onto the column equilibrated in 100 mm sodium phosphate, pH 7.5, and run at room temperature at a flow rate of 2 ml/min, collecting 3-ml fractions. The low molecular weight Gel Filtration Kit (bovine serum albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A) (Amersham Biosciences) served as molecular mass standard. The lactoyltrypanothione thioesters were prepared in two steps. For the generation of mono-(lactoyl)trypanothione, 5 mm trypanothione disulfide in 1 ml of 100 mm MOPS, pH 7.2, was reduced with 7 mm NADPH and 2.75 units of T. cruzi trypanothione reductase for 1 h at room temperature. Then 10 μl of 100 mm methylglyoxal in 100 mm MOPS, pH 7.2, and 6.4 units of yeast glyoxalase I were added and the reaction mixture was incubated for another 1 h. For the synthesis of bis-(lactoyl)trypanothione, 1.5 ml of 3 mm reduced trypanothione was mixed with 8 μl of 5.5 m methylglyoxal and treated as described above. NADPH, methylglyoxal, and the proteins were removed from the thioesters (and excess trypanothione in the case of mono-(lactoyl)trypanothione) on an Oasis® MCX cartridge (Waters). The cartridge was pre-equilibrated with 1 ml of methanol and 2 ml of H2O and the reaction mixture was applied. After washing with 2 ml of H2O and 2 ml of methanol, the thioesters were eluted with 2 ml of 1 m N-ethylmorpholino acetate, pH 6.8. The flow-through already containing some thioester was again applied onto the re-equilibrated cartridge, washed, and eluted with 1 ml of 1 m N-ethylmorpholino acetate, pH 6.8. The thioester containing fractions were dried by evaporation and stored in aliquots at -20 °C. Immediately before use, an aliquot was dissolved in 150 μl of 100 mm MOPS, pH 7.2. The concentration was determined by measuring the thiol content with DTNB before and after hydrolysis of the thioester by glyoxalase II (11Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar). The S-lactoyltrypanothione thioesters eluted from the Oasis MCX cartridge were analyzed by HPLC using a buffer system described in Ref. 12Shahi S.K. Krauth-Siegel R.L. Clayton C.E. Mol. Microbiol. 2002; 43: 1129-1138Crossref PubMed Scopus (80) Google Scholar. After injection of the sample, the column (Vydac 218TP52) was washed at a flow rate of 0.3 ml/min for 5 min with solvent A (0.25% (w/v) d-camphor sulfonate Li-salt, pH 2.64) at 40 °C. Then the thioesters were eluted by a 19-min isocratic step of 90% solvent A and 10% solvent B (25% 1-propanol in solvent A). Before applying a new sample, the column was washed with 100% solvent B for 10 min and equilibrated for 23 min with 100% solvent A. The S-lactoyltrypanothione conjugates were detected by the thioester absorption at 240 nm. To show the regeneration of trypanothione in the T. brucei glyoxalase II reaction, the samples were analyzed at 220 nm. The MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) analyses of the thioesters were carried out by Drs. Jens Pfannstiel and Johannes Lechner (Biochemie-Zentrum der Universität Heidelberg) using a 2,5-dihydroxybenzoic acid matrix. The activities of T. brucei and bovine liver glyoxalase II were determined by measuring the hydrolysis of the thioesters (lactoylglutathione, mono-(lactoyl)trypanothione, and bis-(lactoyl)trypanothione) directly at 240 nm or in a coupled assay following the reaction of the liberated thiol with DTNB at 412 nm (13Ridderström M. Saccucci F. Hellman U. Bergman T. Principato G. Mannervik B. J. Biol. Chem. 1996; 271: 319-323Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The assays were performed in a total volume of 1 ml of 100 mm MOPS, pH 7.2, at 25 °C in a Hitachi 150-20 spectrophotometer or in 300-μl microcuvettes in a Beckman DU®-65 spectrophotometer. The lactoylglutathione concentration was varied between 0.01 and 5 mm in the coupled and between 0.01 and 0.6 mm in the direct assay. Hydrolysis of the lactoyltrypanothione thioesters was followed only in the direct assay at 240 nm at concentrations between 0.02 and 0.15 mm. The thioester stock solutions were freshly prepared from a dried aliquot stored at -20 °C (see above). The extinction coefficients used are DTNB, ϵ412 = 13.6 mm-1 cm-1 (11Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar), SLG, ϵ240 = 3.3 mm-1 cm-1 (14Racker E. J. Biol. Chem. 1951; 190: 685-696Abstract Full Text PDF PubMed Google Scholar, 15Uotila L. Biochemistry. 1973; 12: 3938-3943Crossref PubMed Scopus (46) Google Scholar), MLT, ϵ240 = 3.3 mm-1 cm-1, and BLT, ϵ240 = 6.5 mm-1 cm-1, respectively. The ϵ values of the trypanothione thioesters were derived from measuring the thiol concentration with DTNB after complete hydrolysis. The pH dependence of T. brucei glyoxalase II was determined by following the activity of the enzyme in the presence of 82 μm bis-(lactoyl)trypanothione in a total volume of 200 μl of 100 mm MOPS between pH 6.0 and 9.2 at 25 °C. To elucidate the optimum of ionic strength, assays were performed at 25 °C with 88 μm bis-(lactoyl) trypanothione in a total volume of 200 μl of 10–250 mm MOPS buffers at a constant pH of 7.2. Production of d-lactate was shown by coupling the T. brucei glyoxalase II reaction to the reaction of d- and l-lactate dehydrogenase, respectively, following formation of NADH. After the glyoxalase II reaction with 87.5 μm bis-(lactoyl)trypanothione in 800 μl of 100 mm Tris-HCl, pH 8.5, had run to completion, 5 mm NAD and 2.75 units of d- or l-lactate dehydrogenase were added and the absorption increase at 340 nm (ϵ340 = 6.22 mm-1 cm-1) was followed at 25 °C (16Gutheil W.G. Anal. Biochem. 1998; 259: 62-67Crossref PubMed Scopus (13) Google Scholar). The metal content of His6- and tag-free T. brucei glyoxalase II was analyzed by Dr. Peter Schramel (GSF-Forschungszentrum, Neuherberg) by ICP-OES (inductively coupled-plasma optical emission spectrometry). For comparison, the concentration of different metals in the buffers before and after the columns used for purification were determined. About 12 mg of T. brucei His6-GLX II in 50 mm MES, pH 6.5, was coupled to 0.7 ml of Affi-Gel 10 (Bio-Rad) overnight at 4 °C as described by the manufacturer. The resin was then washed with 50 mm MES, pH 6.5, and the remaining reactive groups were blocked with 0.7 ml of 1 m ethanolamine acetate, pH 8.0, for 1 h at 4 °C. The column was washed 2 times each with 3 ml of PBS, containing 250 mm NaCl, 3 ml of 100 mm glycine, pH 2.5, 3 ml of PBS with 500 mm NaCl, and again with 3 ml of PBS containing 250 mm NaCl. The IgGs from 30 ml of rabbit polyclonal antiserum against T. brucei His6-GLX II (Eurogentec) were precipitated by 33% (NH4)2SO4 overnight at 4 °C. After centrifugation for 20 min at 33,000 × g, the pellet was dissolved in 10 ml of PBS containing 250 mm NaCl, and the solution was applied onto the Affi-Gel 10-His6-GLX II column at room temperature. The flow-through was again applied, and the column was washed with 7 ml of PBS containing 250 mm NaCl. After additional washing with 5 ml of PBS with 500 mm NaCl, the antibodies were eluted with 100 mm glycine, pH 2.5, collecting 1-ml fractions in Eppendorf cups containing 60 μl of 1 m Tris, pH 9.5. The protein concentration was determined at 280 nm. Antibody containing fractions were pooled (3 ml), glycerol was added at 50% saturation, and aliquots were stored at -20 °C. Recombinant T. brucei GLX II and cell lysates of bloodstream and procyclic T. brucei were mixed with 4× SDS loading buffer (250 mm Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.004% bromphenol blue, containing 100 mm Tris(2-carboxyethyl)phosphine solution, neutral pH (Bond-Breaker®, Pierce)) and boiled for 5 min. The proteins were separated by polyacrylamide gel electrophoresis on a 10% SDS gel and transferred onto a Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences) by a Bio-Rad Mini Trans Blot, at 4 °C for 2 h at 150 mA. The membrane was treated with blocking solution (5% milk powder, 1× Tris-buffered saline, 0.05% Tween) overnight at 4 °C. The blot was washed at room temperature three times for 5 min with 1× Tris-buffered saline, 0.05% Tween and then incubated for 1 h with the purified T. brucei GLX II antibodies (see above, dilution 1:500). The membrane was again washed as described above and treated with the secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz, dilution 1:20,000)) for 1 h. After washing with Tris-buffered saline/Tween, the blots were developed using the SuperSignal® West Pico chemiluminescent substrate (Pierce) with exposition times from 30 s to 60 min. Cloning and Structural Comparison of T. brucei Glyoxalase II with Glyoxalases II from Other Species—Blast searches with human glyoxalase II as template revealed two sets of putative glyoxalase II sequences in the T. brucei genome. The deduced protein sequences encoded on chromosomes IV and VI were only 25% identical to each other but showed 30 and 36% identity with human glyoxalase II, respectively. Based on this information, the gene on chromosome VI was cloned from genomic DNA of strain TREU 927/4 (10Melville S.E. Leech V. Gerrard C.S. Tait A. Blackwell J.M. Mol. Biochem. Parasitol. 1998; 94: 155-173Crossref PubMed Scopus (94) Google Scholar). The complete coding region was amplified with two gene-specific primers and sequenced in both directions. PCR on cDNA from bloodstream T. brucei with a sequence-specific primer and a poly(dT) primer amplified a fragment containing the 3′ end of the coding sequence followed by a 390-bp long 3′-untranslated region (data not shown). The deduced protein sequence consists of 296 amino acid residues and clearly classifies the T. brucei protein as glyoxalase II. The highest degree of similarity is found with putative proteins from T. cruzi and L. major where 66 and 51%, respectively, of all residues are identical. In the functionally characterized human and Arabidopsis thaliana glyoxalases II 36 and 31%, respectively, of all residues are conserved. The sequence of a probable hydroxyacylglutathione hydrolase from E. coli shows 30% identical residues (Fig. 1). The isoelectric point (pI) of T. brucei glyoxalase II calculated from the protein sequence is 6.0–6.5, which is comparable with those of the proteins from A. thaliana (6.2) (17Ridderström M. Mannervik B. Biochem. J. 1997; 322: 449-454Crossref PubMed Scopus (40) Google Scholar) and Candida albicans (6.0) (18Talesa V. Rosi G. Bistoni F. Marconi P. Norton S.J. Principato G.B. Biochem. Int. 1990; 21: 397-403PubMed Google Scholar) determined by isoelectric focusing. The putative other kinetoplastid glyoxalases II (Fig. 1) also have theoretical acidic pI values. In general, plant glyoxalases II are acidic proteins with pI values ranging from 4.7 to 6.2, whereas animal enzymes have basic pI values. Isoelectric focusing of recombinant human glyoxalase II and the enzyme isolated from erythrocytes yielded isoelectric points of 8.5 (13Ridderström M. Saccucci F. Hellman U. Bergman T. Principato G. Mannervik B. J. Biol. Chem. 1996; 271: 319-323Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and 8.3 (19Allen R.E. Lo T.W. Thornalley P.J. Eur. J. Biochem. 1993; 213: 1261-1267Crossref PubMed Scopus (39) Google Scholar), respectively. Several glyoxalases II such as the enzyme from Aloe vera (20Norton S.J. Talesa V. Yuan W.J. Principato G.B. Biochem. Int. 1990; 22: 411-418Crossref PubMed Scopus (16) Google Scholar), spinach leaves (21Talesa V. Rosi G. Contenti S. Mangiabene C. Lupattelli M. Norton S.J. Giovannini E. Principato G.B. Biochem. Int. 1990; 22: 1115-1120PubMed Google Scholar), and bovine liver mitochondria (22Talesa V. Principato G.B. Norton S.J. Contenti S. Mangiabene C. Rosi G. Biochem. Int. 1990; 20: 53-58PubMed Google Scholar) show multiple protein bands when subjected to isoelectric focusing. At least some of the bands may be explained by a varying content of their metal cofactors that obviously exchange or get lost easily (see below). Because the molecular and kinetic properties of the known glyoxalases II are very similar, the diversities of their isoelectric points probably reflect the evolutionary distance rather than functional differences of the enzymes. Glyoxalases II contain the highly conserved metal binding motif THXHXDH (23Zang T.M. Hollman D.A. Crawford P.A. Crowder M.W. Makaroff C.A. J. Biol. Chem. 2001; 276: 4788-4795Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In total, five His and two Asp residues interact directly with two metal ions as shown in the three-dimensional structure of human glyoxalase II (24Cameron A.D. Ridderström M. Olin B. Mannervik B. Struct. Fold. Des. 1999; 7: 1067-1078Abstract Full Text Full Text PDF Scopus (153) Google Scholar). All these residues are conserved in the trypanosomatid proteins, suggesting that the parasite proteins also possess metal cofactors (Fig. 1). The structure of human glyoxalase II in complex with a substrate analogue revealed three conserved basic residues that are involved in the fixation of the thioester in the active site. Arg-249, Lys-252, and Lys-143 (numbering of human glyoxalase II) are in close proximity to the glycine carboxylate of the glutathione moiety of the substrate analogue (24Cameron A.D. Ridderström M. Olin B. Mannervik B. Struct. Fold. Des. 1999; 7: 1067-1078Abstract Full Text Full Text PDF Scopus (153) Google Scholar). These residues, present in all glyoxalases II studied so far, are not conserved in the proteins from T. brucei and the other kinetoplastid organisms. This was the first indication that glutathione thioesters are probably not the physiological substrates of the parasite glyoxalases II. Overexpression and Purification of T. brucei Glyoxalase II— The glyoxalase II gene was overexpressed from pQE vectors with and without the N-terminal His6 tag. Purification of His-tagged T. brucei glyoxalase II by metal affinity chromatography yielded 10 mg of pure protein from a 1-liter bacterial culture. The tag-free recombinant protein was purified also in a single step using Q-Sepharose as cation exchanger. About 16 mg of pure protein were obtained from a 1-liter culture. Gel filtration of the recombinant protein on Superdex 75 yielded a relative molecular mass of about 25,000 in accordance with a monomeric structure. This corresponds to all glyoxalases II studied so far, e.g. from A. vera (20Norton S.J. Talesa V. Yuan W.J. Principato G.B. Biochem. Int. 1990; 22: 411-418Crossref PubMed Scopus (16) Google Scholar), human red blood cells (19Allen R.E. Lo T.W. Thornalley P.J. Eur. J. Biochem. 1993; 213: 1261-1267Crossref PubMed Scopus (39) Google Scholar), human liver (13Ridderström M. Saccucci F. Hellman U. Bergman T. Principato G. Mannervik B. J. Biol. Chem. 1996; 271: 319-323Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and A. thaliana (17Ridderström M. Mannervik B. Biochem. J. 1997; 322: 449-454Crossref PubMed Scopus (40) Google Scholar). The molecular mass calculated from the protein sequence is 32,507 in good agreement with 32,000 estimated by SDS-polyacrylamide gel electrophoresis. Synthesis and Structural Analysis of S-Lactoyltrypanothione—Mono- and bis-(lactoyl)trypanothione thioesters were obtained by reacting reduced trypanothione with methylglyoxal. The spontaneous reaction resulted in a hemithioacetal (14Racker E. J. Biol. Chem. 1951; 190: 685-696Abstract Full Text PDF PubMed Google Scholar, 25Rae C. O'Donoghue S.I. Bubb W.A. Kuchel P.W. Biochemistry. 1994; 33: 3548-3559Crossref PubMed Scopus (18) Google Scholar), which is isomerized into the lactoyltrypanothione thioester by yeast glyoxalase I. The method takes advantage of the fact that glyoxalase I, in contrast to glyoxalase II, is not highly specific for glutathione but also accepts derivatives of glutathione modified at the glycine carboxylate (26Cameron A.D. Olin B. Ridderström M. Mannervik B. Jones T.A. EMBO J. 1997; 16: 3386-3395Crossref PubMed Scopus (214) Google Scholar, 27Hamilton D.S. Creighton D.J. Biochim. Biophys. Acta. 1992; 1159: 203-208Crossref PubMed Scopus (8) Google Scholar). The thioesters were freed from the other reaction components by chromatography on an Oasis MCX cation exchanger cartridge that retains the positively charged trypanothione derivatives. HPLC analysis of bis-(lactoyl)trypanothione showed two peaks with retention times of 17.5 and 19.5 min. The first, minor peak represents mono-(lactoyl)trypanothione and the second, major peak is bis-(lactoyl)trypanothione (Fig. 2A). Mass spectrometry confirmed bis-(lactoyl)trypanothione as the main reaction product and the presence of a small amount of mono-(lactoyl)trypanothione (Fig. 2B). Mono-(lactoyl)trypanothione (≥90% pure) was obtained by a" @default.
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• W2018736093 title "Glyoxalase II of African Trypanosomes Is Trypanothione-dependent" @default.
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