| 1. |
- Banerjee, Rahul, et al.
(author)
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Ferritin-Like Proteins : A Conserved Core for a Myriad of Enzyme Complexes
- 2022
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In: Macromolecular Protein Complexes IV. - Cham : Springer. - 9783031007927 - 9783031007934 ; , s. 109-153
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Book chapter (peer-reviewed)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.
(author)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
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In: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Journal article (peer-reviewed)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. |
- Fuller, Franklin D, et al.
(author)
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Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
- 2017
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In: Nature Methods. - : Macmillan Publishers Ltd.. - 1548-7091 .- 1548-7105. ; 14, s. 443-449
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Journal article (peer-reviewed)abstract
- X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
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| 4. |
- John, Juliane, et al.
(author)
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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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In: eLIFE. - 2050-084X. ; 11
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Journal article (peer-reviewed)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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| 5. |
- John, Juliane, et al.
(author)
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Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
- 2022
-
In: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 11
-
Journal article (peer-reviewed)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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| 6. |
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| 7. |
- Lebrette, Hugo, 1986-, et al.
(author)
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Structure of a ribonucleotide reductase R2 protein radical
- 2023
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In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6666, s. 109-113
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Journal article (peer-reviewed)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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| 8. |
- Perálvarez-Marín, Alex, et al.
(author)
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Metal utilization in genome-reduced bacteria : Do human mycoplasmas rely on iron?
- 2021
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In: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 19, s. 5752-5761
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Research review (peer-reviewed)abstract
- Mycoplasmas are parasitic bacteria with streamlined genomes and complex nutritional requirements. Although iron is vital for almost all organisms, its utilization by mycoplasmas is controversial. Despite its minimalist nature, mycoplasmas can survive and persist within the host, where iron availability is rigorously restricted through nutritional immunity. In this review, we describe the putative iron-enzymes, transporters, and metalloregulators of four relevant human mycoplasmas. This work brings in light critical differences in the mycoplasma-iron interplay. Mycoplasma penetrans, the species with the largest genome (1.36 Mb), shows a more classic repertoire of iron-related proteins, including different enzymes using iron-sulfur clusters as well as iron storage and transport systems. In contrast, the iron requirement is less apparent in the three species with markedly reduced genomes, Mycoplasma genitalium (0.58 Mb), Mycoplasma hominis (0.67 Mb) and Mycoplasma pneumoniae (0.82 Mb), as they exhibit only a few proteins possibly involved in iron homeostasis. The multiple facets of iron metabolism in mycoplasmas illustrate the remarkable evolutive potential of these minimal organisms when facing nutritional immunity and question the dependence of several human-infecting species for iron. Collectively, our data contribute to better understand the unique biology and infective strategies of these successful pathogens.
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| 9. |
- Shilova, Anastasiia, et al.
(author)
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Current status and future opportunities for serial crystallography at MAX IV Laboratory
- 2020
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In: Journal of Synchrotron Radiation. - Chichester : Wiley-Blackwell. - 0909-0495 .- 1600-5775. ; 27, s. 1095-1102
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Journal article (peer-reviewed)abstract
- Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
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| 10. |
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