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Title: Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

Abstract

The M. tuberculosis prephenate dehydratase was cloned, expressed, purified, crystallized by the hanging-drop vapour-diffusion method, and a complete data set collected to 3.2 Å resolution using synchrotron radiation. These results should pave the way for the three-dimensional structure determination of the enzyme and provide a framework on which to base the rational design of chemotherapeutic agents to treat tuberculosis. Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 Å resolution using a synchrotron-radiation source.

Authors:
 [1];  [2];  [3];  [4];  [2]; ; ;  [5];  [2]
  1. Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)
  2. (Brazil)
  3. Programa de Pós Graduação em Biofísica Molecular, Departamento de Física, IBILCE/UNESP, São José do Rio Preto, SP, 15054-000 (Brazil)
  4. Programa de Pós Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)
  5. Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS (Brazil)
Publication Date:
OSTI Identifier:
22356352
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 4; Other Information: PMCID: PMC2222585; PMID: 16582484; PUBLISHER-ID: za5129; OAI: oai:pubmedcentral.nih.gov:2222585; 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; CRYSTALS; DESIGN; DIFFUSION; MOLECULES; RESOLUTION; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION

Citation Formats

Vivan, Ana Luiza, Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Dias, Márcio Vinícius Bertacini, Schneider, Cristopher Z., Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Azevedo, Walter Filgueira Jr de, Basso, Luiz Augusto, E-mail: luiz.basso@pucrs.br, Santos, Diógenes Santiago, E-mail: luiz.basso@pucrs.br, and Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106006385.
Vivan, Ana Luiza, Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Dias, Márcio Vinícius Bertacini, Schneider, Cristopher Z., Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Azevedo, Walter Filgueira Jr de, Basso, Luiz Augusto, E-mail: luiz.basso@pucrs.br, Santos, Diógenes Santiago, E-mail: luiz.basso@pucrs.br, & Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv. United Kingdom. doi:10.1107/S1744309106006385.
Vivan, Ana Luiza, Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Dias, Márcio Vinícius Bertacini, Schneider, Cristopher Z., Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS, Azevedo, Walter Filgueira Jr de, Basso, Luiz Augusto, E-mail: luiz.basso@pucrs.br, Santos, Diógenes Santiago, E-mail: luiz.basso@pucrs.br, and Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. Sat . "Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv". United Kingdom. doi:10.1107/S1744309106006385.
@article{osti_22356352,
title = {Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv},
author = {Vivan, Ana Luiza and Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS and Dias, Márcio Vinícius Bertacini and Schneider, Cristopher Z. and Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS and Azevedo, Walter Filgueira Jr de and Basso, Luiz Augusto, E-mail: luiz.basso@pucrs.br and Santos, Diógenes Santiago, E-mail: luiz.basso@pucrs.br and Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS},
abstractNote = {The M. tuberculosis prephenate dehydratase was cloned, expressed, purified, crystallized by the hanging-drop vapour-diffusion method, and a complete data set collected to 3.2 Å resolution using synchrotron radiation. These results should pave the way for the three-dimensional structure determination of the enzyme and provide a framework on which to base the rational design of chemotherapeutic agents to treat tuberculosis. Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 Å resolution using a synchrotron-radiation source.},
doi = {10.1107/S1744309106006385},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 62,
place = {United Kingdom},
year = {Sat Apr 01 00:00:00 EST 2006},
month = {Sat Apr 01 00:00:00 EST 2006}
}
  • Rv2780, an alanine dehydrogenase from M. tuberculosis, has been crystallized in apo and NAD/pyruvate-bound forms. Preliminary crystallographic analysis shows that there is a hexamer and trimer in the asymmetric units of the apo and ternary complex forms, respectively. Rv2780, an alanine dehydrogenase from Mycobacterium tuberculosis (MtAlaDH), catalyzes the NAD-dependent interconversion of alanine and pyruvate. Alanine dehydrogenase is released into the culture medium in substantial amounts by virulent strains of mycobacteria and is not found in the vaccine strain of tuberculosis. Crystals of recombinant MtAlaDH were grown from 2 M ammonium sulfate solution at ∼12 mg ml{sup −1} protein concentration inmore » two crystal forms which occur in the presence and absence of NAD/pyruvate, respectively. Diffraction data extending to 2.6 Å were collected at room temperature from both apo and ternary complex crystals. Crystals of the apoenzyme have unit-cell parameters a = 173.89, b = 127.07, c = 135.95 Å. They are rod-like in shape and belong to space group C2. They contain a hexamer in the asymmetric unit. Crystals of the ternary complex belong to space group P4{sub 3}2{sub 1}2 and have unit-cell parameters a = b = 88.99, c = 373.85 Å. There are three subunits in the asymmetric unit of the holoenzyme crystals.« less
  • A sulfotransferase from M. tuberculosis was crystallized and preliminarily analyzed using X-ray diffraction. Sulfotransferase STF1 from the Mycobacterium tuberculosis H37Rv genome was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 1.5 Å resolution using synchrotron radiation at SPring-8. The crystals are monoclinic and belong to space group P2{sub 1}, with unit-cell parameters a = 40.86, b = 95.76, c = 48.04 Å, β = 106.43°. The calculated Matthews coefficient is approximately 2.1 Å{sup 3} Da{sup −1} assuming the presence of one molecule of STF1 in the asymmetric unit. A substrate-binding assay usingmore » a PAP–agarose column suggests that STF1 exhibits sulfotransferase activity.« less
  • Fatty acid-CoA racemase from M. tuberculosis H37Rv has been overexpressed, purified and crystallized. Diffraction data have been collected to beyond 2.7 Å resolution using a synchrotron-radiation source. Fatty acid-CoA racemase plays an important role in the β-oxidation of branched-chain fatty acids and fatty-acid derivatives as it catalyzes the conversion of several (2R)-branched-chain fatty acid-CoAs to their (2S)-stereoisomers. Fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv has been purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 4000 as precipitant. The crystals belong to the trigonal space group P3{sub 1} or P3{sub 2}, with unit-cell parameters a =more » b = 109.56, c = 147.97 Å. The asymmetric unit contains six monomers, corresponding to a V{sub M} value of 2.15 Å{sup 3} Da{sup −1}. A complete native data set has been collected at 2.7 Å resolution using a synchrotron-radiation source.« less
  • The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2{sub 1}2{submore » 1}2{sub 1}, with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å.« less
  • 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.