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Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-5467-5570
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-5574-9383
Number of Authors: 32019 (English)In: Communications Biology, ISSN 2399-3642, Vol. 2, article id 175Article in journal (Refereed) Published
Abstract [en]

Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 angstrom), in complex with Mn2+ and citrate (1.9 angstrom), and with the CDP-DAG substrate (1.8 angstrom). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism-including a substrate-induced carboxylate shift-for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.

Place, publisher, year, edition, pages
2019. Vol. 2, article id 175
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-169249DOI: 10.1038/s42003-019-0427-1ISI: 000467215700005PubMedID: 31098408OAI: oai:DiVA.org:su-169249DiVA, id: diva2:1327271
Available from: 2019-06-19 Created: 2019-06-19 Last updated: 2020-02-17Bibliographically approved
In thesis
1. Structural basis for metalloprotein catalysis: Characterization of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase PgsA1 and Bacillus anthracis ribonucleotide reductase R2
Open this publication in new window or tab >>Structural basis for metalloprotein catalysis: Characterization of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase PgsA1 and Bacillus anthracis ribonucleotide reductase R2
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

About a third of all proteins need to associate with a particular metal ion or metallo-inorganic cofactor to function. This interplay expands the catalytic repertoire of enzymes and reflects the adaption of these catalytic macromolecules to the environments they have evolved in. A large portion of this work focuses on the membrane metalloprotein PgsA1 from the pathogen Mycobacterium tuberculosis and a radical-harboring protein R2 from the pathogen Bacillus anthracis, offering a glimpse into the metalloprotein universe and the catalysis they perform.

This thesis is divided into two parts; the first part describes a method for high-throughput M. tuberculosis membrane protein expression screening in Escherichia coli and Mycobacterium smegmatis. This method employs target membrane protein fusions with the folding reporter Green Fluorescent Protein, allowing for fast selection of well-expressing membrane protein targets for further structural and functional characterization. This technique allowed overexpression of M. tuberculosis phosphatidylinositol phosphate synthase PgsA1, leading to its crystallization and the characterization of its high-resolution three-dimensional structure. PgsA1 is a MgII- dependent enzyme, catalyzing a vital step in the biosynthesis of phosphatidylinositol – one of the major phospholipids comprising the complex mycobacterial cell envelope. Therefore, PgsA1 presents an attractive target for the development of new antibiotics against tuberculosis.

The second part of this thesis concerns the structural characterization of the B. anthracis class Ib ribonucleotide reductase radical-generating subunit R2 (R2b). R2b contains a dinuclear metallocofactor, which is able to be activated by dioxygen and generates a stable tyrosyl radical; the radical is further used for initiation of nucleotide reduction in the catalytic subunit of ribonucleotide reductase. R2b proteins utilize a di-manganese cofactor in vivo, but can also generate the radical using a di-iron cofactor in vitro, albeit less efficiently. How does R2b achieve correct metallation for efficient catalysis? We show that the B. anthracis R2b protein scaffold is able to select manganese over iron, and furthermore, describe the structural features that govern this metal-specificity. In addition, we describe redox-dependent structural changes in di-iron B. anthracis R2b after reaction with O2, and propose their role in gating solvent access to the metallocofactor and the radical site.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 70
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-178861 (URN)978-91-7911-044-4 (ISBN)978-91-7911-045-1 (ISBN)
Public defence
2020-03-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.

Available from: 2020-03-04 Created: 2020-02-16 Last updated: 2020-05-22Bibliographically approved

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