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Fighting Tuberculosis –: Structural Studies of Three Mycobacterial Proteins
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB).

The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism.

The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel.

The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket , 2008. , p. 94
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 567
Keywords [en]
Mycobacterium tuberculosis, Rv0216, Rv0130, Mycobacterium smegmatis, branched chain aminotransferase, X-ray crystallography, fatty acid metabolism
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-9348ISBN: 978-91-554-7325-9 (print)OAI: oai:DiVA.org:uu-9348DiVA, id: diva2:172754
Public defence
2008-11-29, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2011-06-10Bibliographically approved
List of papers
1. Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial
Open this publication in new window or tab >>Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-97709 (URN)
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2010-01-13Bibliographically approved
2. Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
Open this publication in new window or tab >>Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
2006 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, no 10, p. 2300-2309Article in journal (Refereed) Published
Abstract [en]

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 angstrom. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k(cat) of 1.1 x 10(2) sec(-1). The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.

Keywords
Rv0130, Mycobacterium tuberculosis, hydratase, hotdog fold, crystal structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-94234 (URN)10.1110/ps.062309306 (DOI)000240851300008 ()16963641 (PubMedID)
Available from: 2006-04-12 Created: 2006-04-12 Last updated: 2017-12-14Bibliographically approved
3. Structural analysis of
Open this publication in new window or tab >>Structural analysis of
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-97711 (URN)
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2010-01-13Bibliographically approved

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