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Title: Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus

Abstract

To elucidate the mechanism of regulation of aspartate kinase, the regulatory subunit (the β subunit of T. thermophilus aspartate kinase) was crystallized in the presence of the inhibitor threonine. Aspartate kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the β subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 Å at a synchrotron source. The crystal belongs to the cubic space group P4{sub 3}32 or P4{sub 1}32, with unit-cell parameters a = b = c = 141.8 Å.

Authors:
; ;  [1];  [1];  [2]
  1. Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)
  2. (Japan)
Publication Date:
OSTI Identifier:
22360262
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 2; Other Information: PMCID: PMC2330127; PMID: 17277448; PUBLISHER-ID: gx5106; OAI: oai:pubmedcentral.nih.gov:2330127; 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; DIFFRACTION; FEEDBACK; SPACE GROUPS

Citation Formats

Yoshida, Ayako, Tomita, Takeo, Kuzuyama, Tomohisa, Nishiyama, Makoto, E-mail: umanis@mail.ecc.u-tokyo.ac.jp, and RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148. Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309106055837.
Yoshida, Ayako, Tomita, Takeo, Kuzuyama, Tomohisa, Nishiyama, Makoto, E-mail: umanis@mail.ecc.u-tokyo.ac.jp, & RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148. Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus. United Kingdom. doi:10.1107/S1744309106055837.
Yoshida, Ayako, Tomita, Takeo, Kuzuyama, Tomohisa, Nishiyama, Makoto, E-mail: umanis@mail.ecc.u-tokyo.ac.jp, and RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148. Thu . "Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus". United Kingdom. doi:10.1107/S1744309106055837.
@article{osti_22360262,
title = {Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus},
author = {Yoshida, Ayako and Tomita, Takeo and Kuzuyama, Tomohisa and Nishiyama, Makoto, E-mail: umanis@mail.ecc.u-tokyo.ac.jp and RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148},
abstractNote = {To elucidate the mechanism of regulation of aspartate kinase, the regulatory subunit (the β subunit of T. thermophilus aspartate kinase) was crystallized in the presence of the inhibitor threonine. Aspartate kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the β subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 Å at a synchrotron source. The crystal belongs to the cubic space group P4{sub 3}32 or P4{sub 1}32, with unit-cell parameters a = b = c = 141.8 Å.},
doi = {10.1107/S1744309106055837},
journal = {Acta Crystallographica. Section F},
number = Pt 2,
volume = 63,
place = {United Kingdom},
year = {Thu Feb 01 00:00:00 EST 2007},
month = {Thu Feb 01 00:00:00 EST 2007}
}
  • 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
  • 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
  • SecDF is a multi-path membrane protein required for efficient protein translocation and integration via translocon. Purification and crystallization of T. thermophilus SecDF have been achieved by exploiting unique crystallization techniques that allowed the collection of a 3.74 Å data set. Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in themore » presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P4{sub 3}2{sub 1}2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.« less
  • 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 belongsmore » 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°.« less
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