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Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, 19905-19916 p.Article in journal (Refereed) Published
Abstract [en]

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn2+, NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC50 value of 80 nM. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

Place, publisher, year, edition, pages
2007. Vol. 282, no 27, 19905-19916 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96293DOI: 10.1074/jbc.M701935200ISI: 000247650600065PubMedID: 17491006OAI: oai:DiVA.org:uu-96293DiVA: diva2:170818
Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Structural and Functional Studies of Peptidyl-prolyl cis-trans isomerase A and 1-deoxy-D-xylulose- 5-phosphate reductoisomerase from Mycobacterium tuberculosis
Open this publication in new window or tab >>Structural and Functional Studies of Peptidyl-prolyl cis-trans isomerase A and 1-deoxy-D-xylulose- 5-phosphate reductoisomerase from Mycobacterium tuberculosis
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent.

In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site.

The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria.

The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 103 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 351
Keyword
Molecular biology, Mycobacterium tuberculosis, Rv0009, peptidyl-prolyl cis-trans isomerase, Rv2870c, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, non-mevalonate pathway, DOXP/MEP pathway, X-ray crystallography, Molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8253 (URN)978-91-554-6985-6 (ISBN)
Public defence
2007-11-09, B41, BMC, Husargatan 3, Uppsala, 13:00 (English)
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Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2009-11-29Bibliographically approved

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Unge, TorstenCarlsson, JensÅqvist, JohanMowbray, Sherry L.

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