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A Trypanothione-Dependent Glyoxalase I with a Prokaryotic Ancestry in Leishmania Major

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A Trypanothione-Dependent Glyoxalase I with a Prokaryotic Ancestry in Leishmania Major A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major Tim J. Vickers, Neil Greig, and Alan H. Fairlamb* Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, DD1 5EH Dundee, Scotland Edited by Anthony Cerami, The Kenneth S. Warren Institute, Kitchawan, NY, and approved July 29, 2004 (received for review April 26, 2004) Glyoxalase I forms part of the glyoxalase pathway that detoxifies to hydrolyze the S-D-lactoylglutathione product of GLO1 to reactive aldehydes such as methylglyoxal, using the spontaneously D-lactate and glutathione (1). GLO2 is not as well characterized formed glutathione hemithioacetal as substrate. All known eu- as GLO1 and is not seen as a target for chemotherapy, because karyotic enzymes contain zinc as their metal cofactor, whereas the the reaction catalyzed by GLO1 is essentially irreversible under Escherichia coli glyoxalase I contains nickel. Database mining and physiological conditions; thus, inhibition of GLO2 would only sequence analysis identified putative glyoxalase I genes in the cause the accumulation or excretion of the nontoxic S-D- eukaryotic human parasites Leishmania major, Leishmania infan- lactoylglutathione (10). Interestingly, GLO1 inhibitors are se- tum, and Trypanosoma cruzi, with highest similarity to the cya- lectively toxic to proliferating cells, possibly as a result of an nobacterial enzymes. Characterization of recombinant L. major inhibition of DNA synthesis produced by the accumulation of glyoxalase I showed it to be unique among the eukaryotic enzymes methylglyoxal (11, 12). GLO1 inhibitors have also been reported in sharing the dependence of the E. coli enzyme on nickel. The to have antimalarial (13) and antitrypanosomal (14) activities. parasite enzyme showed little activity with glutathione hemithio- New antiparasitic drugs are required for the treatment of acetal substrates but was 200-fold more active with hemithioac- tropical diseases caused by the pathogenic trypanosomes and etals formed from the unique trypanosomatid thiol trypanothione. Leishmania. These protozoa cause Chagas’ disease, sleeping L. major glyoxalase I also was insensitive to glutathione derivatives sickness, and the various forms of leishmaniasis, which cause that are potent inhibitors of all other characterized glyoxalase I serious illness and death, particularly in many tropical regions. enzymes. This substrate specificity is distinct from that of the The currently available treatments, on the whole, are poor and human enzyme and is reflected in the modification in the L. major suffer from serious side effects, limited efficacy, and both natural sequence of a region of the human protein that interacts with the and acquired resistance (15). New drugs and drug targets are glycyl-carboxyl moiety of glutathione, a group that is conjugated therefore urgently required (16). Such a target may be offered to spermidine in trypanothione. This trypanothione-dependent by the glyoxalase system of the pathogenic kinetoplastids, as a glyoxalase I is therefore an attractive focus for additional biochem- consequence of these protozoa possessing an unusual thiol ical and genetic investigation as a possible target for rational drug metabolism. In these organisms, instead of glutathione, the 1 8 design. major low molecular mass thiol is trypanothione [N ,N - bis(glutathionyl)spermidine] (17). Previous investigations on methylglyoxal ͉ D-lactate ͉ glutathione ͉ nickel Leishmania donovani showed that intact cells quantitatively converted methylglyoxal to D-lactate, suggesting the presence of an active glyoxalase system (18). However, only very low GLO1 ll organisms require systems to shield them from chemical and GLO2 activities could be detected in extracts using gluta- Astress, such as the antioxidant enzymes that detoxify en- thione as a cofactor (19). These observations prompted us to dogenous oxidants and the enzymes that metabolize exogenous ␣ investigate whether trypanothione would substitute for glutathi- toxins. However, endogenous toxins such as the reactive -oxoal- one in the glyoxalase pathway. Here we report the cloning, dehyde methylglyoxal (2-oxopropanal) are also by-products of expression, and characterization of a Leishmania major GLO1 metabolism (1). The main source of methylglyoxal is the non- that is similar to prokaryotic glyoxalases but unique in its enzymatic fragmentation of triose phosphates, but it also is pronounced specificity for trypanothione hemithioacetal over formed during threonine catabolism and acetone oxidation (2). glutathione hemithioacetal as substrate. L. major GLO1 thus In some prokaryotes, dihydroxyacetone phosphate is enzymat- represents a member of a previously uncharacterized family of ically converted to methylglyoxal and inorganic phosphate by GLO1 enzymes. During our investigations into L. major GLO1, methylglyoxal synthase (EC 4.2.3.3) in response to phosphate an independent study reported that GLO2 from Trypanosoma starvation (2). Methylglyoxal reacts rapidly with both proteins brucei was highly selective for mono- and bis-S-D-lactoyltrypano- and nucleic acids and thus is both toxic and mutagenic (3, 4). thione (20). Taken together, these observations provide com- The glyoxalase system is a ubiquitous detoxification pathway pelling evidence for trypanosomatids possessing a unique that protects against the cellular damage caused by methylg- trypanothione-dependent glyoxalase system. lyoxal. This pathway comprises glyoxalase I (GLO1) (lactoylglu- tathione lyase, EC 4.4.1.5) and glyoxalase II (GLO2) (hydroxya- Materials and Methods cylglutathione hydrolase, EC 3.1.2.6), which act in concert to Materials. Saccharomyces cerevisiae GLO1 was purchased from convert the spontaneously formed hemithioacetal adduct be- Sigma. Methylglyoxal was prepared from methylglyoxaldimethy- tween glutathione (␥-L-glutamyl-L-cysteinylglycine) and meth- ylglyoxal into D-lactate and free glutathione (1). Thus, glutathi- one acts as a cofactor in the overall reaction pathway. GLO1 is This paper was submitted directly (Track II) to the PNAS office. a metalloenzyme that isomerizes the glutathione hemithioacetal Freely available online through the PNAS open access option. to S-D-lactoylglutathione through proton transfer to a metal- Abbreviations: GLO1, glyoxalase I; GLO2, glyoxalase II; trypanothione, N1,N8-bis(glutathio- bound enediol intermediate (5). This metal cofactor is zinc in nyl)spermidine. eukaryotes but nickel in Escherichia coli (6, 7). The difference in Data deposition: The sequence reported in this paper (Leishmania major GLO1 gene) has cofactor dependence is reflected in differences between the been deposited in the GenBank database (accession no. AY604654). active sites of the human and E. coli enzymes, suggesting that the *To whom correspondence should be addressed. E-mail: [email protected]. latter may be a target for antimicrobial therapy (8, 9). GLO2 acts © 2004 by The National Academy of Sciences of the USA 13186–13191 ͉ PNAS ͉ September 7, 2004 ͉ vol. 101 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402918101 Downloaded by guest on September 30, 2021 lacetal and confirmed to be free of formaldehyde, as described pKa of the protein was measured by chromatofocusing with a in ref. 21. S-p-bromobenzylglutathione was synthesized as de- MonoPHR5͞20 column (Amersham Pharmacia) and a pH scribed in ref. 22. All other reagents were standard commercial 7.0–4.0 gradient of Polybuffer 74. products of high purity. Production and Assay of Thiols. Trypanothione disulphide (2.5 Determination of Dissociation Constants. Methylglyoxal stocks were ␮mol; Bachem) in 0.5 ml of 400 mM Hepes buffer, pH 7.4, was assayed by preparation of the disemicarbazone adduct (23) and reduced by the addition of a 0.5-ml suspension of Tris(2- ␮ with S. cerevisiae GLO1 (24), both giving identical values. The Kd carboxyethyl)phosphine agarose (containing 4.8 mol of reduc- values for the adducts between glutathione or trypanothione and tant; Pierce). After mixing (25°C for 30 min), the agarose was phenylglyoxal were determined as described in ref. 24. However, removed, HCl was added to 20 mM, and the acidified solution the extinction coefficient of the trypanothione-phenylglyoxal was stored at Ϫ20°C. Thiol concentrations were determined by adduct was determined by using excess phenylglyoxal rather than titration with 5,5Ј-dithio-bis(2-nitrobenzoic acid) (26) by using excess trypanothione, because the use of millimolar levels of an extinction coefficient of 14.14 mMϪ1⅐cmϪ1 at 412 nm for the trypanothione is impractically expensive. A dual-beam Shi- thionitrobenzoate anion (27). madzu UV-2401 PC spectrophotometer was used with a blank assay lacking thiol in the reference beam. The measured extinc- Enzyme Kinetics. Kinetic constants were determined at 27°Cin tion coefficients then were used to determine the concentration 0.5-ml assays containing 100 mM (Naϩ) phosphate, pH 7.0. To of adduct in a set of equilibrium mixtures in 100 mM (Naϩ) ensure full saturation with cofactor, GLO1 was diluted with ␮ ͞ phosphate, pH 7.0. The concentrations used (expressed as [SH assay buffer containing 20 M NiCl2 and 0.02% (wt vol) BSA. group]) were 100 ␮M phenylglyoxal plus 100 ␮M thiol, 100 ␮M The required concentration of hemithioacetal and 0.1 mM free phenylglyoxal plus 200 ␮M thiol, and 200 ␮M phenylglyoxal plus thiol were produced by varying the thiol and aldehyde concen- 100 ␮M thiol. trations, with the concentrations
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