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Title: Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

Abstract

An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.

Authors:
 [1];  [2];  [1]
  1. Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)
  2. Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)
Publication Date:
OSTI Identifier:
22360241
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 1; Other Information: PMCID: PMC2330099; PMID: 17183164; PUBLISHER-ID: bo5010; OAI: oai:pubmedcentral.nih.gov:2330099; 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; CONVERSION; CRYSTALLIZATION; CRYSTALS; PROTEINS; RESOLUTION; ROTATION; SOLVENTS; SPACE GROUPS; SYMMETRY; SYNCHROTRON RADIATION; SYNTHESIS; X-RAY DIFFRACTION

Citation Formats

Bagautdinov, Bagautdin, Sugahara, Mitsuaki, and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309106051578.
Bagautdinov, Bagautdin, Sugahara, Mitsuaki, & Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3. United Kingdom. doi:10.1107/S1744309106051578.
Bagautdinov, Bagautdin, Sugahara, Mitsuaki, and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp. Mon . "Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3". United Kingdom. doi:10.1107/S1744309106051578.
@article{osti_22360241,
title = {Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3},
author = {Bagautdinov, Bagautdin and Sugahara, Mitsuaki and Kunishima, Naoki, E-mail: kunisima@spring8.or.jp},
abstractNote = {An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.},
doi = {10.1107/S1744309106051578},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 63,
place = {United Kingdom},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The archaeal phosphoglycerate mutase PH0037 from P. horikoshii OT3 has been crystallized in space group R32, with unit-cell parameters a = 155.62, c = 230.35 Å. A 2.2 Å resolution data was collected at SPring-8 beamline BL26B1. Phosphoglycerate mutases catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways. The archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to a resolution of 2.2 Å has been collected and processed in space group R32. Assuming themore » presence of a dimer in the asymmetric unit, the V{sub M} value is calculated to be 3.0 Å{sup 3} Da{sup −1}, consistent with the dynamic light-scattering experiment result, which shows a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of Bacilllus stearothermophilus phosphoglycerate mutase as a search model did not provide a satisfactory solution, indicating substantially different structures of these two phophoglycerate mutases.« less
  • The biotin–protein ligase from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with biotin, ADP and biotinyl-5′-AMP. The crystals belong to space group P2{sub 1} and diffract to beyond 1.6 Å resolution.
  • The E subunit of vacuole-type ATPase from P. horikoshii OT3 was overexpressed, purified and crystallized. The native crystals diffracted X-rays to 1.85 Å resolution. The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an R{submore » merge} of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit.« less
  • RecA superfamily ATPase PH0284 from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with ATP. Both crystal forms belong to the trigonal space group P3{sub 2}21 and diffract X-rays to 2.0 and 2.3 Å resolution, respectively. Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 Å resolution using synchrotronmore » radiation at 100 K. The crystal belongs to the trigonal space group P3{sub 2}21, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V{sub M} value of 2.36 Å{sup 3} Da{sup −1} and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 Å resolution.« less
  • The putative thiamine-biosynthesis protein PH1313 from P. horikoshii OT3 was overexpressed, purified and crystallized. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1} and diffract X-rays to 1.9 Å resolution. The putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 has been overexpressed and purified. Crystallization was performed by the oil-microbatch method using 28%(v/v) 2-methyl-2,4-pentanediol as a precipitant at 291 K. A native X-ray diffraction data set at 1.9 Å resolution and a single anomalous dispersion data set from a selenomethionine-derivative crystal at 2.1 Å resolution were collected using synchrotron radiation at 100 K. The native crystal belongs to themore » orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 71.7, b = 71.2, c = 141.8 Å.« less