Glyoxalase II of African Trypanosomes Is Trypanothione-dependent

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. 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 (1, 2). 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 (3). 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) 1 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 (N 1 ,N 8 -bis(glutathionyl)spermidine) and the flavoenzyme trypanothione reductase (4,5). A linkage between glutathione and spermidine metabolism was first discovered in Escherichia coli (6). 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 (7) and is involved...

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

“…Although the L. major genome contains a putative GLO2 (LMFLCHR12 92), which is 51% identical to T. brucei GLO2 at the amino acid level (20), we were unable to detect any GLO2 activity in cell extracts of L. major, which may be because of the insensitivity of the assay method or instability of the enzyme. T. brucei GLO2 readily loses activity because of loss of its metal cofactor (20), and thus L. major GLO2 may have lost activity during dialysis to remove trypanothione and glutathione cosubstrates.…”

“…L. major GLO1 thus represents a member of a previously uncharacterized family of GLO1 enzymes. During our investigations into L. major GLO1, an independent study reported that GLO2 from Trypanosoma brucei was highly selective for mono-and bis-S-D-lactoyltrypanothione (20). Taken together, these observations provide compelling evidence for trypanosomatids possessing a unique trypanothione-dependent glyoxalase system.…”

“…T. brucei GLO2 readily loses activity because of loss of its metal cofactor (20), and thus L. major GLO2 may have lost activity during dialysis to remove trypanothione and glutathione cosubstrates. BLAST searches of the T. cruzi genome identified homologues of GLO1 (Tc00.1047053510659.240 and Tc00.1047053510743.70) and GLO2 (Tc00.1047053507603.230 and Tc00.1047053509429.290).…”

“…Trypanothione-dependent GLO2 activity was determined at 27°C in 0.5 ml of 100 mM Mops, (pH 7.2)͞150 M trypanothione-SH͞200 M methylglyoxal (20). Lactoyltrypanothione was produced by adding 0.3 nmol of recombinant L. major GLO1, and the reaction was monitored at 240 nm.…”

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

“…Uniquely, this is dependent upon trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine or T[SH] 2 ) 2 (4), whereas their human hosts use glutathione ((␥-L-glutamyl-L-cysteinylglycine or GSH). Trypanothione is involved in protective processes, defending against oxidative stress through peroxidase systems (5), against reactive aldehydes through the glyoxalase system (6,7), and against toxic xenobiotics through the trypanothione S-transferase (TST) (8). All these processes depend upon trypanothione being maintained in its dithiol form by trypanothione reductase.…”