Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme

Abstract

The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization.1. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%.2. The enzyme is a dimer with a subunit M , of 50000. Using NADPH (K, = 5 pM) and trypanothione disulfide (K, = 45 pM) as substrates, a turnover number of 14200 min-' was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates.3. When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay.4. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions E,, + NADPH + H' + EH2 + NADP', and EH, + trypanothione disulfide + E,, + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH&S04. 5. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electronreduced form (EH,) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfide at the catalytic site. After reduction of E,, to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.Trypanosomes and leishmanias are unicellular eukaryotes which belong to the family Trypanosomatidue and to the order Kinetoplastida [l, 21. Diseases of man and domestic animals caused by these protozoa include sleeping sickness (Trypanosoma rhodesiense, T . gambiense), Chagas' disease ( T . cruzi), nagana (T. brucei, T . vivax, T . congolense), oriental sore (Leishmania tropica) and kala-azar (L. donovani). Most parasitic Kinetoplastida are thought to be more sensitive to oxidative stress than their hosts; indeed recent evidence suggests that the host's defense system and the action of certain drugs is largely based on reactive oxygen species [3 -51. In addition, there is a qualitative difference: the major antioxidant enzyme system of the parasites needs trypanothione [6] and possibly other glutathionylspermidines Abbreviations. E,,, enzyme in oxidized form; EH2, two-electronreduced enzyme; GSH, reduced glutathione; GSSG, glutathione disulfide; Nbsz, 5,5'-dithiobis(2-nitrobenzoate), Ellman's reagent; trypanothione disulfide, N1,N8-bis(L-y-glutamyl-L-hemicystinylglycy1)spermidine; dihydrotrypanothione, N' ,N8-bis(L-y-glutamyl-Lcysteinyl-glycy1)spermidine.

Mechanisms of flavoprotein-catalyzed reactions

“…The enzyme has been isolated from Crithidiu fusciculatu [86] and Trypunosomu cruzi [87] and shown to have very similar properties to those of glutathione reductase, including molecular mass, non-covalently bound FAD, an active-site disulfide with 14 residues identical with glutathione reductase, the formation of a typical EH2 charge-transfer spectrum and specific alkylation of EH2 with iodoacetamide at the active-site cysteine closest to the N-terminus [86, 871. While little structural or mechanistic work has yet been reported, the reaction mechanism is undoubtedly similar to that of glutathione reductase.…”

Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

“…1). In these organisms, T(S)2 plays an important role in the maintenance of reduced thiols and is itself maintained in its reduced state by the enzyme trypanothione reductase (TR; CAS registry number 102210-35-5) (10,11). GR and TR are members of a family of enzymes, the flavoprotein disulfide oxidoreductases, and possess similar kinetic properties.…”

“…GR and TR are members of a family of enzymes, the flavoprotein disulfide oxidoreductases, and possess similar kinetic properties. However, they have almost mutually exclusive substrate specificities (10)(11)(12)(13). The gene encoding TR in Trypanosoma congolense has been isolated and overexpressed (14,15), and alignment of the inferred amino acid sequence with the primary structures of human (16) and E. coli (17) GRs has shown 41% and 38% sequence identity, respectively.…”

<i>IN SILICO</i> AND <i>IN VITRO</i> STUDIES: TRYPAREDOXIN PEROXIDASE INHIBITOR ACTIVITY OF METHOTREXATE FOR ANTILEISHMANIAL ACTIVITY

“…This enzyme cascade involves Trypanothione Reductase (Try R), Tryparedoxin (TXN) and trypanotione (N 1 , N 8 -bis (glutathionyl)-spermidine) serving as a mediator for Science Publications AJID transfer of reducing equivalents Shames et al, 1986). The first enzyme of the cascade is homologous to glutathione reductase and thioredoxin reductase (Krauth-Siegel et al, 1987) which is involved in NADPH-dependent hydroperoxide reduction in other species (Tamura et al, 1995). The other components of the trypanosomatid system also belong to protein families occasionally constituting peroxidase systems.…”