Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata

Massimo Degano, Deshmukh N. Gopaul, Giovanna Scapin, Vern L. Schramm, James C. Sacchettini

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 Å. The enzyme has an open (α,β) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded β sheet surrounded by six α helices and a small C-terminal lobe composed of four α helices. Two short antiparallel β strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of β strands β1 and β4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid-base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262].

Original languageEnglish
Pages (from-to)5971-5981
Number of pages11
JournalBiochemistry
Volume35
Issue number19
Publication statusPublished - May 14 1996

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N-Glycosyl Hydrolases
Crithidia fasciculata
Inosine
Uridine
Biochemistry
Catalysis
Catalytic Domain
Uracil-DNA Glycosidase
Purine-Nucleoside Phosphorylase
Salvaging
Purines
Ribose
X ray crystallography
X Ray Crystallography
Enzyme Inhibitors
Substrates
Nucleosides
Histidine
Aspartic Acid
Hydroxyl Radical

ASJC Scopus subject areas

  • Biochemistry

Cite this

Degano, M., Gopaul, D. N., Scapin, G., Schramm, V. L., & Sacchettini, J. C. (1996). Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. Biochemistry, 35(19), 5971-5981.

Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. / Degano, Massimo; Gopaul, Deshmukh N.; Scapin, Giovanna; Schramm, Vern L.; Sacchettini, James C.

In: Biochemistry, Vol. 35, No. 19, 14.05.1996, p. 5971-5981.

Research output: Contribution to journalArticle

Degano, M, Gopaul, DN, Scapin, G, Schramm, VL & Sacchettini, JC 1996, 'Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata', Biochemistry, vol. 35, no. 19, pp. 5971-5981.
Degano M, Gopaul DN, Scapin G, Schramm VL, Sacchettini JC. Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. Biochemistry. 1996 May 14;35(19):5971-5981.
Degano, Massimo ; Gopaul, Deshmukh N. ; Scapin, Giovanna ; Schramm, Vern L. ; Sacchettini, James C. / Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. In: Biochemistry. 1996 ; Vol. 35, No. 19. pp. 5971-5981.
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abstract = "Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 {\AA}. The enzyme has an open (α,β) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded β sheet surrounded by six α helices and a small C-terminal lobe composed of four α helices. Two short antiparallel β strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of β strands β1 and β4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid-base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupi{\~n}{\'a}n, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262].",
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