The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins.

Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.

Crystallization of recombinant Crithidia fasciculata tryparedoxin.

Recombinant tryparedoxin, a thioredoxin homologue from Crithidia fasciculata, has been purified from an Escherichia coli expression system and used in crystallization trials. Orthorhombic needles in space group P212121, with unit cell dimensions of a = 38.63, b = 51. 47, and c = 73.41 A, have been obtained. The crystals present a monomer of approximate molecular mass 16 kDa in the asymmetric unit and diffract to 1.8-A resolution using synchrotron radiation. Structure determination will be carried out to further the understanding of the role tryparedoxin plays in regulating oxidative stress in parasitic trypanosomatids.

Targeting metabolic pathways in microbial pathogens: oxidative stress and anti-folate drug resistance in trypanosomatids.

The large quantity of genomic, biochemical and metabolic data on microbial pathogens provides information that helps us to select biological problems of interest and to identify targets, metabolic pathways or constituent enzymes, for therapeutic intervention. One area of potential use in developing novel anti-parasitic agents concerns the regulation of oxidative stress, and we have targeted the trypanothione peroxidase pathway in this respect. In order to characterize this pathway, we have determined crystal structures for each of its components, and are now studying enzyme-ligand complexes of the first enzyme, trypanothione reductase. Also with regard to trypanosomatids, a question that arose was: why do anti-folates not provide useful therapies? The enzyme pteridine reductase has been shown to contribute to anti-folate drug resistance, and we have determined the enzyme structure and mechanism to understand this aspect of drug resistance.

Structure of the macrocycle thiostrepton solved using the anomalous dispersion contribution of sulfur.

The structure of a tetragonal crystal form of thiostrepton has been solved using the anomalous dispersive effects of five S atoms from high-redundancy data collected to 1.33 A resolution at the Cu Kalpha wavelength. Data measured to 1.02 A resolution with a synchrotron source were used for refinement. Details of the molecular structure, intramolecular and intermolecular interactions are given.

The high resolution crystal structure of recombinant Crithidia fasciculata tryparedoxin-I.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.