Publications

• Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis.

  Versees W., Barlow J., Steyaert J.

  Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase wi ... >> More

  Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity. << Less

  J Mol Biol 359:331-346(2006) [PubMed] [EuropePMC]

• Purification, characterization, and gene cloning of purine nucleosidase from Ochrobactrum anthropi.

  Ogawa J., Takeda S., Xie S.X., Hatanaka H., Ashikari T., Amachi T., Shimizu S.

  A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversibl ... >> More

  A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K(m) values being 11.8 to 56.3 microM. The optimal activity temperature and pH were 50 degrees C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K(i) and K(i)' values of 0.455 to 11.2 microM. Metal ion chelators inhibited activity, and the addition of Zn(2+) or Co(2+) restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH(2)-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases. << Less

  Appl Environ Microbiol 67:1783-1787(2001) [PubMed] [EuropePMC]

• Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei. Purification, specificity, and kinetic mechanism.

  Parkin D.W.

  Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Tr ... >> More

  Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Trypanosoma brucei brucei represents approximately 0.2% of the soluble protein in this organism. The enzyme has been purified over 400-fold to >95% homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of Mr 36,000 subunits. The kcat/Km for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x 10(6), and 0.83 x 10(6) M -1 s-1, respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degrees C is 34 s-1. Pyrimidine nucleosides are poor substrates with kcat/Km values of approximately 10(3) M -1 s-1. Deoxynucleosides are also poor substrates with kcat/Km values near 10(2) M -1 s-1. AMP is not a detectable substrate and there is no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin), and formycin B are competitive inhibitors with Kis of 1.8, 59, and 13 microM, respectively. The Km shows a slight dependence on pH with a pH optimum around 7. The Vmax/Km data indicate there are two ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The Vmax data indicate three ionizable groups involved in catalysis. Two essential groups exhibit pKa values of 8.8, and a third group with a pKa of 6.5 increases the Vmax an additional 10-fold. All three groups must be protonated for optimum catalytic activity. << Less

  J Biol Chem 271:21713-21719(1996) [PubMed] [EuropePMC]

• Enzyme-substrate interactions in the purine-specific nucleoside hydrolase from Trypanosoma vivax.

  Versees W., Decanniere K., Van Holsbeke E., Devroede N., Steyaert J.

  Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (). Here we repo ... >> More

  Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (). Here we report the 2.0-A crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-A crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2'-OH, 3'-OH, and 5'-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k(cat)/K(m), respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp(83) and Trp(260). The pH profiles of k(cat) and k(cat)/K(m) indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation. << Less

  J Biol Chem 277:15938-15946(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Examination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from Trypanosoma vivax.

  Barlow J.N., Steyaert J.

  The mechanism and energetics of the purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) are examined by stopped-flow at low temperatures. TvNH is shown to follow an ordered uni-bi kinetic mechanism and high forward commitment with inosine as substrate (C(f) = 1.9 +/-0.6). Measuremen ... >> More

  The mechanism and energetics of the purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) are examined by stopped-flow at low temperatures. TvNH is shown to follow an ordered uni-bi kinetic mechanism and high forward commitment with inosine as substrate (C(f) = 1.9 +/-0.6). Measurement of partitioning of the Michaelis complex, which exists at negligible concentrations in the steady state, is achieved using a novel sequential-mixing stopped-flow method. A product burst is observed with p-nitrophenyl riboside (pNPR) in the pre-steady state, indicating that a step after chemistry rate determines k(cat). Comparison of the kinetics of inosine and pNPR turnover shows that the dominant energetic contribution towards catalysis in TvNH comes from ribosyl and water activation (11 kcal/mol); however, leaving group activation still makes a considerable (8 kcal/mol) contribution. A solvent isotope effect ((D2O)k = 1.7) on the chemistry transient tau1 with guanosine as substrate was observed. Therefore, the leaving group is unlikely to be protonated prior to N-glycosidic bond cleavage. We propose that leaving group protonation is, by itself, unlikely to account for the large energetic contribution of leaving group activation. Instead, we postulate that active site binding interactions to the purine leaving group are required for efficient ribosyl and/or water activation. << Less

  Biochim Biophys Acta 1774:1451-1461(2007) [PubMed] [EuropePMC]