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Structural and multimerization dynamics of Mycobacterium tuberculosis Fatty acyl CoA synthetase FadD13
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.ORCID-id: 0000-0001-8854-6937
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism.

FadD13 is a peripheral membrane protein, with both soluble and membrane-bound populations in vivo. The protein displays a prominent positively charged surface patch, suggested to be involved in membrane association. Here we characterize the lipid binding properties of FadD13 and further show that the protein adopts a dimeric arrangement in solution. The dimer interface partly buries the positive patch, seemingly inconsistent with membrane binding. Moreover, the dimer arrangement does not provide an obvious alternative mode of membrane interaction.

To gain further insight into the membrane binding, two protein variants were created, one where the positive patch was altered to become more negative and one more hydrophobic. The hydrophobic variant displayed an apparent increase in lipid affinity and the negative variant still retained significant lipid binding. Structural analysis showed that dimerization was disrupted in the variant proteins, with both variants being predominantly monomeric in solution, thus exposing the proposed membrane-binding surface. Together, the results suggest that FadD13 membrane interaction is regulated by a dimer-monomer equilibrium.

Nyckelord [en]
acetyl coenzyme A (acetyl‐CoA), acetyl‐CoA synthetase, fatty acid metabolism, fatty acid binding protein, Mycobacterium tuberculosis, dimerization, membrane protein, membrane enzyme, small‐angle X‐ray scattering (SAXS)
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-167811OAI: oai:DiVA.org:su-167811DiVA, id: diva2:1302349
Tillgänglig från: 2019-04-04 Skapad: 2019-04-04 Senast uppdaterad: 2019-04-04Bibliografiskt granskad
Ingår i avhandling
1. Structural and Functional Studies of Membrane Proteins: From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase
Öppna denna publikation i ny flik eller fönster >>Structural and Functional Studies of Membrane Proteins: From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis is divided into three parts; the first part describes a method for efficient screening of membrane proteins for crystallography. By utilising the properties of a folding reporter GFP it is possible to quickly and accurately screen the stability of a protein in a range of conditions without full purification. This allows rapid assessment of the suitability of a protein for crystallography and a parallel optimisation of purification conditions for subsequent large-scale protein production.

The second part describes the discovery of a membrane bound superoxide oxidase (SOO), a novel scavenger of membrane proximal superoxide. SOO is a kinetically perfect enzyme, reacting at rates close to the diffusion limit in a similar fashion to other superoxide scavengers, such as superoxide dismutase. We propose that SOO rescues electrons “lost” to superoxide and recycles them back into the respiratory chain, releasing oxygen. At the same time SOO contributes to the proton motive force by uptake of protons from the cytoplasmic side of the membrane.

The third part concerns the fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis (M. tuberculosis). It represents a critical node point in M. tuberculosis lipid metabolism and has been suggested to be a vital component of M. tuberculosis survival in host cell macrophages. FadD13 harbours a hydrophobic cavity that is unable to house the very-long-chain substrates the enzyme has preference for. We propose that FadD13 is a peripheral membrane protein, utilising the membrane to house the very-long-chain fatty acid substrates during the activation reaction.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 74
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-167812 (URN)978-91-7797-648-6 (ISBN)978-91-7797-649-3 (ISBN)
Disputation
2019-05-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Tillgänglig från: 2019-05-06 Skapad: 2019-04-04 Senast uppdaterad: 2019-05-02Bibliografiskt granskad

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Lundgren, CamillaHögbom, Martin
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Institutionen för biokemi och biofysik
Biokemi och molekylärbiologi

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