Structures of Tryparedoxins Revealing Interaction with Trypanothione

170

Barley 2-cysteine peroxiredoxin (2-Cys Prx) was analyzed for peroxide reduction, quaternary structure, thylakoid attachment, and function as well as in vivo occurrence of the inactivated form, with emphasis on the role of specific amino acid residues. Data presented show the following. 1) 2-Cys Prx has a broad substrate specificity and reduces even complex lipid peroxides such as phosphatidylcholine dilineoyl hydroperoxide, although at low rates. 2) 2-Cys Prx partly becomes irreversibly oxidized by peroxide substrates during the catalytic cycle in a concentration-dependent manner, particularly by bulky hydroperoxides.

Section: Structural Changes In 2-cys Prx Based On Fluorescence Of Sinmentioning

confidence: 85%

Section: Sequence Alignment Characterizes 2-cys Prx As a Hydrophobic mentioning

confidence: 99%

Reaction Mechanism of Plant 2-Cys Peroxiredoxin

König¹,

Lotte

Plessow

et al. 2003

170

“…The carboxyl groups of GSH, Gsp, and T(SH) 2 are typically bound to arginines and less often to lysines (50), as has been discussed for glutathione peroxidases (51,52), glutathione reductase (53), tryparedoxin (54), and trypanothione reductase (55), for example. The very same residues are also implicated in binding and activating ATP.…”

Section: Discussionmentioning

confidence: 99%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

Self Cite

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 (GenBank TM 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 M, 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.

“…The crystal structures of C. fasciculata tryparedoxin revealed that the first active site cysteine is also located at the N-terminal end of an ␣-helix protruding out of the protein molecule (16,17) and that there are no basic groups that might promote dissociation of the nucleophilic cysteine. Activation of the first cysteine is suggested to be achieved by the second cysteine, whose proton appears to be loosened by a network of hydrogen bonds (17).…”

Section: Discussionmentioning

confidence: 99%

“…The three-dimensional structure of C. fasciculata tryparedoxin revealed a folding very similar to that of thioredoxins into a five-stranded ␤-sheet surrounded by four ␣-helices (16,17). In particular the active site motif Cys 40 -Pro 41 -Pro 42 -Cys 43 is in a position homologous to that of the corresponding motif in thioredoxin.…”

mentioning

confidence: 96%

Catalytic Properties, Thiol p K Value, and Redox Potential of Trypanosoma brucei Tryparedoxin

Reckenfelderbäumer

Krauth‐Siegel

2002

The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of ؊249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/ tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys 40 , the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to <4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.