David W. Parkin

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei.

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial import sequence at its N-terminus and has a variant IPC motif replacing the second VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cytoplasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are expressed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclonal antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibody to TRYP2 were generated in mice. In-situ immunofluorescence and immunoelectron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol, whereas TRYP2 was principally localised in the mitochondrion.

Molecular Cloning and Expression of a Purine-specific N-Ribohydrolase from Trypanosoma brucei brucei SEQUENCE, EXPRESSION, AND MOLECULAR ANALYSIS

N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size-polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, theiagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense,Trypanosoma evansi, Trypanosoma congolense, andTrypanosoma vivax and, to a lesser extent, withTrypanosoma cruzi genomic DNA. The iagnh gene is expressed in bloodstream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme fromCrithidia fasciculata. The T. b. brucei iagnhopen reading frame was cloned into Escherichia coliBL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

Enzymatic transition states and inhibitor design from principles of classical and quantum chemistry

rn A procedure is described which leads to experimentally based models for the transitionstate structures of enzyme-catalyzed reactions. Substrates for an enzymic reaction are synthesized with isotopically enriched atoms at every position in which bonding changes are anticipated at the enzyme-enforced transition state. Kinetic isotope effects are measured for each atomic substitution and corrected for diminution of the isotope effects from nonchemical steps of the enzymic mechanism. A truncated geometric model of the transition-state structure is fitted to the kinetic isotope effects using bond-energy bondorder vibrational analysis. Full molecularity is restored to the transition state while maintaining the geometry of the bonds which define the transition state. Electronic wave functions are calculated for the substrate and the transition-state molecules. The molecular electrostatic potential energies are defined for the van der Waal surfaces of substrate and transition state and displayed in numerical and color-coded constructs. The electronic differences between substrate and transition state reveal characteristics of the transition state which permits the extraordinary binding affinity of enzyme-transition state interactions. The information has been used to characterize several enzymatic transition states and to design powerfully inhibitory transition-state analogues. Enzymatic examples are provided for the reactions catalyzed by AMP deaminase, nucleoside hydrolase, purine nucleoside phosphorylase, and for several bacterial toxins. The results demonstrate that the combination of experimental, classical, and quantum chemistry approaches is