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

Title: Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum

Abstract

Dihydropyrimidinase from the slime mould D. discoideum was crystallized. A single crystal was shown to belong to space group I222 and diffracted anisotropically to better than 1.8 Å. Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated β-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 Å resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 Å and one molecule in the asymmetric unit.

Authors:
 [1]; ;  [2];  [1]
  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm (Sweden)
  2. Department of Cell and Organism Biology, Lund (Sweden)
Publication Date:
OSTI Identifier:
22356238
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 1; Other Information: PMCID: PMC2150923; PMID: 16511257; PUBLISHER-ID: en5141; OAI: oai:pubmedcentral.nih.gov:2150923; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; DIFFUSION; MOLECULES; MONOCRYSTALS; RESOLUTION; SPACE GROUPS; X-RAY DIFFRACTION

Citation Formats

Lohkamp, Bernhard, Andersen, Birgit, Piškur, Jure, and Dobritzsch, Doreen, E-mail: doreen.dobritzsch@ki.se. Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum. United Kingdom: N. p., 2006. Web. doi:10.1107/S174430910503976X.
Lohkamp, Bernhard, Andersen, Birgit, Piškur, Jure, & Dobritzsch, Doreen, E-mail: doreen.dobritzsch@ki.se. Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum. United Kingdom. doi:10.1107/S174430910503976X.
Lohkamp, Bernhard, Andersen, Birgit, Piškur, Jure, and Dobritzsch, Doreen, E-mail: doreen.dobritzsch@ki.se. Sun . "Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum". United Kingdom. doi:10.1107/S174430910503976X.
@article{osti_22356238,
title = {Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum},
author = {Lohkamp, Bernhard and Andersen, Birgit and Piškur, Jure and Dobritzsch, Doreen, E-mail: doreen.dobritzsch@ki.se},
abstractNote = {Dihydropyrimidinase from the slime mould D. discoideum was crystallized. A single crystal was shown to belong to space group I222 and diffracted anisotropically to better than 1.8 Å. Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated β-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 Å resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 Å and one molecule in the asymmetric unit.},
doi = {10.1107/S174430910503976X},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 62,
place = {United Kingdom},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • No abstract prepared.
  • Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.
  • M. tuberculosis diaminopimelate decarboxylase, the enzyme that catalyzes the final step of lysine biosynthesis, has been cloned, expressed, purified and crystallized in the absence of cofactor or substrate. Diaminopimelate decarboxylase from Mycobacterium tuberculosis (LysA, DAPDC, Rv1293) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized. Preliminary diffraction data analysis suggests the presence of a homotetramer in the asymmetric unit.
  • Propionate kinase (TdcD) from S. typhimurium has been expressed, purified and crystallized. A diffraction data set has been collected to 2.2 Å resolution. In the cell, propionate is mainly formed during β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that l-threonine is non-oxidatively cleaved to propionate via 2-ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.–). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonellamore » typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 111.47, c = 66.52 Å. A complete data set to 2.2 Å resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.« less
  • M. tuberculosis dihydrodipicolinate reductase, the enzyme that catalyzes the second committed step of lysine biosynthesis, has been cloned, expressed, purified and crystallized in three different crystal forms. Dihydrodipicolinate reductase from Mycobacterium tuberculosis (DapB, DHDPR, Rv2773c) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in three different crystal forms. Preliminary diffraction data analysis suggests the presence of two tetramers in the asymmetric unit of one crystal form and half a tetramer in the other two crystal forms.