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1.  Structure of Trypanosoma brucei glutathione synthetase: Domain and loop alterations in the catalytic cycle of a highly conserved enzyme 
Graphical abstract
The close similarity of Trypanosoma brucei glutathione synthetase to the human orthologue indicates that the enzyme would be a difficult target for drug discovery.
Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-γ-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery.
doi:10.1016/j.molbiopara.2009.12.011
PMCID: PMC2845819  PMID: 20045436
AMP-PNP, adenylyl imidodiphosphate; GS, glutathione synthetase; GSH, glutathione; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid); MOPS, 3-(N-morpholino)-propanesulfonic acid; NCS, non-crystallographic symmetry; Tb, Trypanosoma brucei; TEV, tobacco etch virus; TLS, translation/libration/screw; TSA, trypanothione synthetase; T[SH]2, trypanothione; ATP-grasp; Glutathione; Glutathione synthetase; Trypanosoma brucei; Trypanothione; X-ray structure
2.  Trypanosoma brucei UDP-galactose-4′-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose 
The structure of recombinant T. brucei UDP-galactose-4′-epimerase cocrystallized with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose has been determined at medium resolution. Comparisons with structures of human and E. coli UDP-galactose-4′-epimerase–ligand complexes reveal that the hexose moieties are able to adopt different orientations in the active site.
The structure of the NAD-dependent oxidoreductase UDP-galactose-4′-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose has been determined using diffraction data to 2.7 Å resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-α-d-galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180° with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site.
doi:10.1107/S1744309106028740
PMCID: PMC2242870  PMID: 16946458
short-chain dehydrogenase/reductases; Trypanosoma brucei; UDP-galactose-4′-epimerase; UDP-4-deoxy-4-fluoro-α-d-galactose
3.  High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei  
Attempts to crystallize a complex of papain (C. papaya) with a cysteine protease inhibitor from the parasitic pathogen T. brucei failed. However, over an extended period the mixture produced an ordered crystal of the protease carrying two peptide fragments in the active site. These correspond to dipeptides and tripeptides that are assigned as fragments of the inhibitor, which has presumably suffered proteolytic cleavage.
Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Comparisons reveal that these peptides bind in the active site in a manner similar to that of the human cysteine protease inhibitor stefin B when it is complexed to papain. The assignment of the fragment sequences is consistent with the specificity of the protease.
doi:10.1107/S1744309106014849
PMCID: PMC2243108  PMID: 16754967
papain; cysteine protease; inhibitors; Trypanosoma brucei

Results 1-3 (3)