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Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. 

To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. 

Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. 

Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2012. , 60 p.
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-75750ISBN: 978-91-7447-512-8 (print)OAI: oai:DiVA.org:su-75750DiVA: diva2:523825
Public defence
2012-06-01, Magnélisalen, Kemiska Övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 3: In press; Paper 4: Manuscript.

Available from: 2012-05-11 Created: 2012-04-26 Last updated: 2012-09-28Bibliographically approved
List of papers
1. A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold
Open this publication in new window or tab >>A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold
2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, no 14, 5633-8 p.Article in journal (Refereed) Published
Abstract [en]

Chlamydia trachomatis R2c is the prototype for a recently discovered group of ribonucleotide reductase R2 proteins that use a heterodinuclear Mn/Fe redox cofactor for radical generation and storage. Here, we show that the Mycobacterium tuberculosis protein Rv0233, an R2 homologue and a potential virulence factor, contains the heterodinuclear manganese/iron-carboxylate cofactor but displays a drastic remodeling of the R2 protein scaffold into a ligand-binding oxidase. The first structural characterization of the heterodinuclear cofactor shows that the site is highly specific for manganese and iron in their respective positions despite a symmetric arrangement of coordinating residues. In this protein scaffold, the Mn/Fe cofactor supports potent 2-electron oxidations as revealed by an unprecedented tyrosine-valine crosslink in the active site. This wolf in sheep's clothing defines a distinct functional group among R2 homologues and may represent a structural and functional counterpart of the evolutionary ancestor of R2s and bacterial multicomponent monooxygenases.

Keyword
bioinorganic chemistry, diiron, manganese, monooxygenase, R2c
National Category
Natural Sciences
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-32232 (URN)10.1073/pnas.0812971106 (DOI)000264967500034 ()19321420 (PubMedID)
Available from: 2009-12-07 Created: 2009-12-07 Last updated: 2012-05-02Bibliographically approved
2. The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.
Open this publication in new window or tab >>The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.
Show others...
2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, 123-125 p.Article in journal (Refereed) Published
Abstract [en]

The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

National Category
Structural Biology
Identifiers
urn:nbn:se:su:diva-75648 (URN)10.1021/ja209678x (DOI)000301084200032 ()22133609 (PubMedID)
Funder
Swedish Research Council, 2010-5200The Wenner-Gren FoundationSwedish Foundation for Strategic Research Swedish Research Council, 2010-5061Swedish Research Council, 2010-4950
Available from: 2012-04-24 Created: 2012-04-24 Last updated: 2017-12-07Bibliographically approved
3. The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme
Open this publication in new window or tab >>The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme
Show others...
2012 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 20, no 6, 1062-1070 p.Article in journal (Refereed) Published
Abstract [en]

The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-79995 (URN)10.1016/j.str.2012.03.012 (DOI)000305094500014 ()
Funder
Swedish Research Council, 2010-5061Swedish Foundation for Strategic Research , FFL4
Note

AuthorCount:7;

Available from: 2012-09-19 Created: 2012-09-12 Last updated: 2017-12-07Bibliographically approved
4. A Dynamic C-terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein can Assume a Helical Structure with Possible Functional Consequences
Open this publication in new window or tab >>A Dynamic C-terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein can Assume a Helical Structure with Possible Functional Consequences
(English)Manuscript (preprint) (Other academic)
Keyword
Structural dynamics, Bioinorganic, ribonucleotide reductase, heterodinuclear, oxidase
National Category
Structural Biology
Identifiers
urn:nbn:se:su:diva-75669 (URN)
Available from: 2012-04-24 Created: 2012-04-24 Last updated: 2013-09-10Bibliographically approved

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