Inosine - uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue

Deshmukh N. Gopaul, Sheryl L. Meyer, Massimo Degano, James C. Sacchettini, Vern L. Schramm

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Abstract

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidiafasciculata is the inosine - uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to >99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in kcat for inosine but a 2.8-fold increase in kcat with p-nitrophenyl β-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 Å resolution, respectively. Both belong to the P21212 orthorhombic space group with unit cell parameters a = 63.5 Å, b = 131.9 Å, c = 90.1 Å, and α = β = γ = 90°. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

Original languageEnglish (US)
Pages (from-to)5963-5970
Number of pages8
JournalBiochemistry
Volume35
Issue number19
StatePublished - May 14 1996

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N-Glycosyl Hydrolases
Crithidia fasciculata
Inosine
Uridine
Crystallization
Histidine
Catalytic Domain
Enzymes
Amino Acids
Escherichia coli
Salvaging
Amino Acid Sequence
Open Reading Frames
Complementary DNA
Protozoa
Leishmania major
Proteins
Escherichia coli Proteins
Polymerase chain reaction
DNA sequences

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inosine - uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue. / Gopaul, Deshmukh N.; Meyer, Sheryl L.; Degano, Massimo; Sacchettini, James C.; Schramm, Vern L.

In: Biochemistry, Vol. 35, No. 19, 14.05.1996, p. 5963-5970.

Research output: Contribution to journalArticle

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abstract = "Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidiafasciculata is the inosine - uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to >99{\%} homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in kcat for inosine but a 2.8-fold increase in kcat with p-nitrophenyl β-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 {\AA} resolution, respectively. Both belong to the P21212 orthorhombic space group with unit cell parameters a = 63.5 {\AA}, b = 131.9 {\AA}, c = 90.1 {\AA}, and α = β = γ = 90°. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.",
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T1 - Inosine - uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue

AU - Gopaul, Deshmukh N.

AU - Meyer, Sheryl L.

AU - Degano, Massimo

AU - Sacchettini, James C.

AU - Schramm, Vern L.

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N2 - Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidiafasciculata is the inosine - uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to >99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in kcat for inosine but a 2.8-fold increase in kcat with p-nitrophenyl β-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 Å resolution, respectively. Both belong to the P21212 orthorhombic space group with unit cell parameters a = 63.5 Å, b = 131.9 Å, c = 90.1 Å, and α = β = γ = 90°. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

AB - Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidiafasciculata is the inosine - uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to >99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in kcat for inosine but a 2.8-fold increase in kcat with p-nitrophenyl β-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 Å resolution, respectively. Both belong to the P21212 orthorhombic space group with unit cell parameters a = 63.5 Å, b = 131.9 Å, c = 90.1 Å, and α = β = γ = 90°. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

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