skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8

Abstract

The pyrimidine nucleoside phosphorylase TTHA1771 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffract X-rays to 1.8 Å resolution using synchrotron radiation. Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure–thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291 K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8 Å resolution using synchrotron radiation. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 58.83, b = 76.23, c = 103.86 Å, β = 91.3°.

Authors:
;  [1]
  1. Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cyo, Sayo-gun, Hyogo 679-5148 (Japan)
Publication Date:
OSTI Identifier:
22360306
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 4; Other Information: PMCID: PMC2330216; PMID: 17401202; PUBLISHER-ID: ll5101; OAI: oai:pubmedcentral.nih.gov:2330216; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; RESOLUTION; SPACE GROUPS; SYNCHROTRON RADIATION; SYNTHESIS; X-RAY DIFFRACTION

Citation Formats

Shimizu, Katsumi, and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107009980.
Shimizu, Katsumi, & Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8. United Kingdom. doi:10.1107/S1744309107009980.
Shimizu, Katsumi, and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Sun . "Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8". United Kingdom. doi:10.1107/S1744309107009980.
@article{osti_22360306,
title = {Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8},
author = {Shimizu, Katsumi and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp},
abstractNote = {The pyrimidine nucleoside phosphorylase TTHA1771 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffract X-rays to 1.8 Å resolution using synchrotron radiation. Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure–thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291 K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8 Å resolution using synchrotron radiation. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 58.83, b = 76.23, c = 103.86 Å, β = 91.3°.},
doi = {10.1107/S1744309107009980},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 63,
place = {United Kingdom},
year = {Sun Apr 01 00:00:00 EDT 2007},
month = {Sun Apr 01 00:00:00 EDT 2007}
}
  • The crystallization and preliminary X-ray diffraction analysis of the l-fuculose-1-phosphate aldolase (FucA) from T. thermophilus HB8. Native diffraction data set was collected to a resolution of 1.9 Å. Fuculose phosphate aldolase catalyzes the reversible cleavage of l-fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 Å. The presence of a dimer ofmore » the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (V{sub M}) of 2.7 Å{sup 3} Da{sup −1} and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 Å from zinc-containing crystals. Native diffraction data to 1.9 Å resolution have been collected using synchrotron radiation at SPring-8.« less
  • The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1} and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation.more » The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.« less
  • The putative fumarylacetoacetase TTHA0809 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffracted X-rays to 2.2 Å resolution using synchrotron radiation. Fumarylacetoacetase catalyzes the final step of tyrosine and phenylalanine catabolism. A recombinant form of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8 has been crystallized by the oil-microbatch method using sodium chloride as a precipitating agent. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.3, b = 73.4, c = 122.6 Å, β = 111.8°. The crystals are most likely to contain two dimers in the asymmetricmore » unit, with a V{sub M} value of 3.32 Å{sup 3} Da{sup −1}. Diffraction data were collected at 2.2 Å resolution using synchrotron radiation at beamline BL26B1 of SPring-8, Japan.« less
  • Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamermore » is the biological active form of E. coli. purine nucleoside phosphorylase.« less
  • Crystals of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from T. thermophilus HB8 were obtained which diffracted synchrotron X-rays to a resolution of 1.6 Å. The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodiummore » acetate pH 5.0 and 25%(v/v) glycerol and diffracted X-rays to a resolution of 1.60 Å. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 Å. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V{sub M} of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 Å. MAD data were successfully collected for structural determination using these crystals.« less