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Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosine
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
2009 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, no Part 6, 618-620 p.Article in journal (Refereed) Published
Abstract [en]

Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni(2+)-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 A resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 A, beta = 124.8 degrees . The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 A(3) Da(-1). Structure determination is in progress.

Place, publisher, year, edition, pages
2009. Vol. 65, no Part 6, 618-620 p.
Keyword [en]
methionyl-tRNA synthetase, MetRS, Mycobacterium tuberculosis, methionine, adenosine
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-110780DOI: 10.1107/S1744309109016704ISI: 000266452100020PubMedID: 19478446OAI: oai:DiVA.org:uu-110780DiVA: diva2:278275
Available from: 2009-11-24 Created: 2009-11-24 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Structural studies of Caseinolytic protease 1 from Mycobacterium tuberculosis and Methionyl-tRNA synthetase from Mycobacterium smegmatis
Open this publication in new window or tab >>Structural studies of Caseinolytic protease 1 from Mycobacterium tuberculosis and Methionyl-tRNA synthetase from Mycobacterium smegmatis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[en]
Abstract [en]

Tuberculosis is a severe disease that causes about 2 million deaths every year. It is a worldwide threat and it is estimated that one-third of the world’s population carries the infection. The severe side effects of the present drugs, and the more than 6 months long treatment, in addition to the development of resistant bacterial strains, are the incentives for the intensified search for new drugs. In this work two potential mycobacterial drug targets have been studied: Caseinolytic protease 1 (ClpP1) from Mycobacterium tuberculosis (Mt) and Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis (Ms).

The X-ray stucture of ClpP1 was determined to 3.0 Å resolution. The study gives details on the tetradecameric arrangement of the enzyme. Two hepameric discs assemble to form a chamber containing the catalytic activity mediated by each of the monomers. The chamber can be reached by two pores. Comparison with the human homologue reveals important structural differences.

The X-ray studies on Ms MetRS were done to 2.3 Å and 2.8 Å resolution. The study gives details on the flexibility of the enzyme and how this is related to activity. Important findings are identification of an intermediate structure in which the methionine to be adenylated is bound in the catalytic site in a tight complex. The catalytic site and the anticodon recognizing domains are separated and the structural results indicate communication between the domains. The possibility to allosterically inhibit the enzyme is discussed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 728
Keyword
Mycobacterium tuberculosis, caseinolytic protease 1, ClpP1, Mycobacterium smegmatis, methionyl-tRNA synthetase, MetRS, X-ray crystallography
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-121779 (URN)978-91-554-7769-1 (ISBN)
Public defence
2010-05-07, B41, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-16 Created: 2010-03-30 Last updated: 2012-09-18

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