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. 2006 Dec 1;400(2):217-23.
doi: 10.1042/BJ20060882.

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

Neil Greig  1 , Susan Wyllie, Tim J Vickers, Alan H Fairlamb
Affiliations

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

Neil Greig et al. Biochem J. .

Abstract

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.

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Figures

Figure 1
Figure 1. Alignment of the predicted amino acid sequences of glyoxalase I
Gaps introduced into sequences to optimize alignments are represented by dashes. Conserved and similar residues are indicated by asterisks and dots respectively. The circles indicate conserved metal binding residues in the various sequences. Squares indicate conserved residues implicated in the binding of γ-glutamate moieties of thiol cofactors while triangles highlight conserved acidic residues (Asp100 and Tyr101) in the trypanosomatid enzymes thought to be involved in the binding of T[SH]2 [28]. Protein sequences are from T. cruzi (Tc00.1047053510659), L. major (LmjF35.3010), E. coli (Swiss-Prot accession no. P77036), Synechoccus sp. WH 8102 (TrEMBL accession no. Q7U3T2), human (Swiss-Prot accession no. P78375) and mouse (Swiss-Prot accession no. Q9CPU0).
Figure 2
Figure 2. Purification of recombinant TcGLO1
Lane 1, soluble fraction of BL21 (DE3)pLysS [pET15b-TcGLO1], uninduced cells; lane 2, soluble fraction of BL21 (DE3)pLysS [pET15b-TcGLO1], induced cells; lane 3, pooled fractions from nickel affinity chromatography; lane 4, pooled fractions from anion exchange chromatography (Resource-Q); lane 5, pooled fractions from anion exchange chromatography (Resource-Q) after removal of the His6-tag with thrombin protease.
Figure 3
Figure 3. Metal reconstitution of TcGLO1 in various bivalent metal chlorides
The apo form of TcGLO1 was incubated with a 10-fold molar excess of bivalent metal ions for 10 min at room temperature. Assays contained 25 μM trypanothione hemithioacetal and 0.1 mM free T[SH]2. L. major GLO1 reconstituted activity (black bars; [11]) is shown next to the TcGLO1 data (white bars). All assays were performed in triplicate, with the mean activity relative to the nickel-activated enzymes displayed.
Figure 4
Figure 4. Enzymatic characterization of TcGLO1
(A) Determination of assay pH optimum. Assays were performed with 50 μM trypanothione hemithioacetal in a mixed buffer system, as described in the Materials and methods section. Activity is expressed as a percentage relative to the maximum activity determined for TcGLO1. Results are presented as means±S.D. from three measurements. (B) Effect of buffer concentration and ionic strength on TcGLO1 activity. The assay mixtures contained either various amounts of Hepes buffer, pH 7.0 (closed circles), NaCl (open circles), or (NH4)2SO4 (open squares). Activity is expressed as a percentage of the activity determined in 50 mM Hepes buffer/25 mM NaCl. The inset shows the effect on activity as a function of buffer or salt concentration.
Figure 5
Figure 5. S-4-bromobenzylglutathionylspermidine inhibition of TcGLO1
TcGLO1 inhibition constants were determined at three fixed concentrations of the inhibitor [(S-4-bromobenzylglutathionylspermidine] and over a range of substrate concentrations (25-125 μM trypanothione hemithioacetal). Data sets for each inhibitor concentration were fitted by non-linear regression and are presented as Lineweaver–Burk transformations. Trypanothione hemithioacetal was varied in the presence of 0 (○), 3.15 (■), 6.3 (□) and 12.6 μM (●) (S)-4-bromobenzylglutathionylspermidine.
Figure 6
Figure 6. Immunoblot analysis of cell lysates and cellular localization of TcGLO1
(A) Blots of whole cell extracts (30 μg of protein in each lane) from T. cruzi epimastigotes (lane 1) and L. major promastigotes (lane 2) were probed with an anti-(L. major GLO1) antiserum. (B) Subcellular fractions of L. major promastigotes, containing the large granule (LG), cytosol (C), and microsomal fractions (MF), were prepared as described in the Materials and methods section. Western blots of these equally loaded fractions (30 μg of protein in each lane) were probed with anti-(L. major GLO1) antibody. In addition, blots were stripped and reprobed with antiserum to marker proteins for each subcellular fraction to demonstrate the purity of each fraction [LG, anti-Hsp60; C, anti-(L. major trypanothione synthetase); and MF, anti-BiP].
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