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The missing piece of the type II Fatty Acid Synthase system from Mycobacterium tuberculosis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2007 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 37, 14628-14633 p.Article in journal (Refereed) Published
Abstract [en]

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C12) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

Place, publisher, year, edition, pages
2007. Vol. 104, no 37, 14628-14633 p.
Keyword [en]
(3R)-hydroxyacyl-ACP dehydratase, hydratase 2, mycolic acid biosynthesis, fatty acid elongation, hot dog fold
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-96919DOI: 10.1073/pnas.0704132104ISI: 000249513000017PubMedID: 17804795OAI: oai:DiVA.org:uu-96919DiVA: diva2:171655
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2011-01-24Bibliographically approved
In thesis
1. Targeting Mycobacterium tuberculosis Proteins: Structure and Function Studies of Five Essential Proteins
Open this publication in new window or tab >>Targeting Mycobacterium tuberculosis Proteins: Structure and Function Studies of Five Essential Proteins
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the target selection, cloning, expression, purification, crystallization, structure and biochemical characterization of five essential Mycobacterium tuberculosis (Mtb) proteins. The search for drugs against the causal agent of tuberculosis is urgently needed and the targeting of essential genes is necessary to fulfill this goal.

The crystal structures of carbonic anhydrases (CA) Rv1284 and Rv3588c have been determined to 2.0 and 1.7 Å resolution, respectively. Rv3588c, in contrast to Rv1284, is an active β-CA that shows two different active site conformations and pH-dependent oligomerization states.

Rv1295 is an active threonine synthase with an unusually high pH optimum; the structure has been solved to 2.5 Å resolution, based on which a modification to the reaction mechanism published previously is proposed.

Mtb has a thick and impermeable cell envelope that constitutes an efficient barrier against drugs. One of the essential components of the envelope is mycolic acid (MA). The inhibition of enzymes participating in its synthesis would be lethal for Mtb. Rv0636, a formerly unknown-function protein has β-hydroxyacyl-ACP dehydrase activity which is essential for MA synthesis. Co-expression with partners notably improves its solubility.

Around 55% of Mtb proteins have unknown function. Rv3778c is one of them and its three-dimensional structure has been determined to 1.8 Å resolution. Studies aimed at the elucidation of its biochemical function are shown. A pathway not yet reported in Mtb is also suggested.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 61 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 411
Cell and molecular biology, carbonic anhydrase, threonine synthase, FAS II, Rv0636, Rv1284, Rv1295, Rv3588c, Rv3778c, X-ray crystallography, Cell- och molekylärbiologi, Mycobacterium tuberculosis
urn:nbn:se:uu:diva-8580 (URN)978-91-554-7134-7 (ISBN)
Public defence
2008-04-11, B42, BMC, Husargatan 3, Uppsala, 13:00 (English)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2009-06-01Bibliographically approved

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