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Structural and functional studies of mycobacterial IspD enzymes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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2011 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, 403-414 p.Article in journal (Refereed) Published
Abstract [en]

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

Place, publisher, year, edition, pages
2011. Vol. 67, 403-414 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-154127DOI: 10.1107/S0907444911006160ISI: 000290235200002PubMedID: 21543842OAI: oai:DiVA.org:uu-154127DiVA: diva2:419292
Available from: 2011-05-26 Created: 2011-05-26 Last updated: 2015-04-23Bibliographically approved
In thesis
1. Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design
Open this publication in new window or tab >>Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria.

The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined.

The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods.

The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 949
Keyword
Tuberculosis, Mycobacterium tuberculosis, MEP pathway, CoA pathway, drug development, crystal structure, DXR, IspD, PanK
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry; Molecular Biology
Identifiers
urn:nbn:se:uu:diva-179057 (URN)978-91-554-8416-3 (ISBN)
Public defence
2012-09-21, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
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Available from: 2012-08-30 Created: 2012-08-06 Last updated: 2013-01-22Bibliographically approved

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