| 1. |
- Banerjee, Rahul, et al.
(författare)
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Ferritin-Like Proteins : A Conserved Core for a Myriad of Enzyme Complexes
- 2022
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Ingår i: Macromolecular Protein Complexes IV. - Cham : Springer. - 9783031007927 - 9783031007934 ; , s. 109-153
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Bokkapitel (refereegranskat)abstract
- Ferritin-like proteins share a common fold, a four α-helix bundle core, often coordinating a pair of metal ions. Although conserved, the ferritin fold permits a diverse set of reactions, and is central in a multitude of macromolecular enzyme complexes. Here, we emphasize this diversity through three members of the ferritin-like superfamily: the soluble methane monooxygenase, the class I ribonucleotide reductase and the aldehyde deformylating oxygenase. They all rely on dinuclear metal cofactors to catalyze different challenging oxygen-dependent reactions through the formation of multi-protein complexes. Recent studies using cryo-electron microscopy, serial femtosecond crystallography at an X-ray free electron laser source, or single-crystal X-ray diffraction, have reported the structures of the active protein complexes, and revealed unprecedented insights into the molecular mechanisms of these three enzymes.
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| 2. |
- Diamanti, Riccardo, et al.
(författare)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
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| 3. |
- John, Juliane, et al.
(författare)
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Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
- 2022
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Ingår i: eLIFE. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
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| 4. |
- Lebrette, Hugo, 1986-, et al.
(författare)
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Structure of a ribonucleotide reductase R2 protein radical
- 2023
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6666, s. 109-113
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Tidskriftsartikel (refereegranskat)abstract
- Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
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| 5. |
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| 6. |
- Srinivas, Vivek, 1988-
(författare)
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To metal, or not to metal : Diverse mechanisms of O2-activation and radical storage in the ferritin superfamily
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins in the Ferritin-like superfamily are characterized by a four alpha-helical structural motif. These proteins are distributed across all three kingdoms of life and perform a wide range of functions. Several members in this protein superfamily can activate dioxygen using a di-metal active site coordinated by four carboxylate and two histidine amino acid residues. The resulting diverse set of dioxygen activated intermediates is used in nature to perform complex redox chemical reaction in cells. The R2 subunit of class I Ribonucleotide reductase and soluble Methane monooxygenase are the most well-characterized groups of proteins in this superfamily. Upon oxygen (or reduced-oxygen) activation of the di-metal site, the R2 subunit can generate a catalytic radical required for the conversion of ribonucleotides to deoxyribonucleotides, while soluble Methane monooxygenase can oxidize methane to methanol in an alternative form of carbon assimilation.The work presented in this thesis aims to better understand metal selectivity, working and the regulation of substrate specificity in various proteins of the Ferritin-like superfamily, and the development of a novel method to study radiation-sensitive intermediates. The papers discussed in this thesis present crystallographic and spectroscopic studies of several Ferritin-like superfamily proteins.In paper I, the assembly mechanisms of the heterodinuclear manganese-iron cofactor in a class Ic R2 protein and an R2-like ligand-binding oxidase are compared. Paper II presents the discovery of a novel radical-generating subunit subclass of Ribonucleotide reductase in Mollicutes, including mycoplasma pathogens, that breaks the paradigm of metal requirement for radical translocation and catalysis. This new subclass, denoted class Ie, is shown to instead use an unprecedented modified tyrosine DOPA residue in its four-helix bundle for radical translocation and storage. Paper III presents a new X-ray free-electron laser sample delivery system that combines acoustic droplet ejection with a drop-on-tape setup, allowing simultaneous multimodal X-ray diffraction and X-ray emission data collection. This setup is also shown to support photochemical and chemical activation of catalysis in crystals, allowing the study of radiation-sensitive transient reaction intermediates. We used this setup in paper IV to solve the first radiation damage-free crystallographic structures of the soluble methane monooxygenase hydroxylase and its regulatory subunit complex from Methylosinus trichosporium OB3b. The high-resolution crystal structures of the complex, in both di-ferrous and di-ferric oxidation states, illustrate the structural reorganization in the hydroxylase subunit upon binding to the regulatory subunit.These results illustrate the functional range and flexibility in the Ferritin-like protein superfamily. Including the distinctive metal discrimination in heterodinuclear metalloproteins, influencing substrate specificity in sMMO, and using a novel metal-free DOPA radical to catalyze ribonucleotide reduction in the class Ie R2 subclass. Experiments using the novel ADE-DOT setup also showed promising progress towards determining the highly sought-after structures of di-metal oxygen activated intermediates such as X and Q in subclass Ia R2 and sMMO, respectively.
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