Trypanothione Synthesis in Crithidia Revisited

Marcelo Comini,Ulrich Menge,Josef Wissing,Leopold Flohé

doi:10.1074/jbc.m404486200

Abstract

In Crithidia fasciculata the biosynthesis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBanktrade mark accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg(2+)-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K(m) values for GSH, Mg(2+)-ATP, and glutathionylspermidine were 407, 222, and 480 microm, respectively, and the k(cat) was 8.7 s(-1) for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.

Highlights

Crithidia fasciculata has been the most convenient model organism to work out the metabolic pathways that are typical of human pathogens such as Trypanosoma and Leishmania species
T(SH)2 has been shown to be of pivotal importance for the vitality and virulence of Trypanosoma brucei brucei and Leishmania donovani by knock-out or knock-down of trypanothione reductase, the enzyme regenerating T(SH)2 from its oxidized cyclic disulfide form [1, 2]
Trypanosomatids are equipped with a typical thioredoxin [12], TXN appears to substitute for thioredoxin in ribonucleotide reduction [13], which may explain the growth arrest observed upon depletion of T(SH)2 [3] or trypanothione reductase [1]

Summary

The abbreviations used are

T(SH), reduced trypanothione; TXN, tryparedoxin; TXN-Px, tryparedoxin peroxidase; TryS, trypanothione synthetase; Gsp, glutathionylspermidine; GspS, glutathionylspermidine synthetase; Ni-NTA, nickel-nitrilotriacetic acid; MALDI-TOF, matrix-assisted laser desorption and ionization time-of-flight; HPLC, high pressure liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RT, room temperature. A GspS devoid of TryS activity was purified from C. fasciculata by Koenig et al [24] and produced by heterologous expression by Oza et al [25].2. These apparent discrepancies between T(SH) biosynthesis of Crithidia and Trypanosoma species raised concerns whether C. fasciculata was an adequate model organism if the ultimate goal is the design of trypanocidal GspS or TryS inhibitors [21]. We report that the putative TryS gene of C. fasciculata encodes a TryS that, like that of the Trypanosoma species, catalyzes both steps of trypanothione synthesis. Over, the yields obtained by heterologous expression of this gene for the first time enabled an in-depth analysis of such prototype of trypanothione synthetase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION