| 1. |
- Aurelius, Oskar, et al.
(författare)
-
The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
- 2015
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
|
|
| 2. |
- Aurelius, Oskar
(författare)
-
To reduce a ribonucleotide – Radical solutions in enzymology in form and function
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- It has been more than 50 years since the enzyme system ribonucleotide reductase (RNR), catalysing the reduction of ribonucleotides to deoxyribonucleotides, was first discovered. RNR was also the first time that radical chemistry was revealed in an enzyme. RNRs carry out a key step in the de novo synthesis of building blocks for DNA and have been found in almost all known organisms. Within this thesis the anaerobic RNR from the thermophilic bacteria Thermotoga maritima has been studied by making use of X-ray crystallography, anaerobic enzyme assays, small-angle X-ray scattering and complementary biophysical methods. The crystal structure of the catalytic subunit of this anaerobic RNR stood out by lacking the typical cysteine residue, which takes part in radical transfer to the substrate, positioned in its active site. The opposing site for the glycyl radical was however structurally conserved, as in structures of other glycyl radical enzymes. It was shown that the glycyl radical could be generated by the addition of the radical generating subunit with a reduced Fe4S4 cluster and the co-substrate S-adenosyl methionine. By also adding T. maritima cell extracts it was verified that the system was an active RNR. Further work focused on understanding complex formation between the two subunits and the structural manifestation of allosteric regulation. Anaerobic analytical ultracentrifugation indicated that a dimer of the catalytic subunit interacts with a monomer of the radical generating subunit. Crystal structures of the catalytic subunit in complex with various nucleotides were used to compare hydrogen bonding networks and nucleotide recognition for allosteric regulation. The second model system studied was the Pseudomonas aeruginosa aerobic RNR catalytic subunit. Proteins from P. aeruginosa are of clinical interest as possible drug targets, due to the many human infections caused by this microorganism. By solving the crystal structure of the catalytic subunit in complex with dATP it became possible to visualise its tetramerisation interface and its surprising capacity to bind three dATP molecules over two ATP cones per monomer. This work shows how RNRs, present in very different types of organisms, still keep on delivering surprises and different solutions to the complexity of synthesising deoxyribonucleotides and regulating nucleotide pools in the cells.
|
|
| 3. |
- Bjelčić, Monika, et al.
(författare)
-
Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes
- 2023
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 79:Pt 11, s. 1018-1025
-
Tidskriftsartikel (refereegranskat)abstract
- In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a 'sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.
|
|
| 4. |
- Bågenholm, Viktoria, et al.
(författare)
-
Galactomannan catabolism conferred by a polysaccharide utilisation locus of Bacteroides ovatus : enzyme synergy and crystal structure of a β-mannanase
- 2017
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 292:1, s. 229-243
-
Tidskriftsartikel (refereegranskat)abstract
- A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two β-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized β-mannanases, we propose a schemeof sequential action by the glycoside hydrolasesencodedby the β-mannanPULandinvolved in the β-mannanutilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the β-mannanase BoMan26A.
|
|
| 5. |
- Fekry, Mostafa, et al.
(författare)
-
The Crystal Structure of Tyrosinase from Verrucomicrobium spinosum Reveals It to Be an Atypical Bacterial Tyrosinase
- 2023
-
Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 13:9
-
Tidskriftsartikel (refereegranskat)abstract
- Tyrosinases belong to the type-III copper enzyme family, which is involved in melanin production in a wide range of organisms. Despite similar overall characteristics and functions, their structures, activities, substrate specificities and regulation vary. The tyrosinase from the bacterium Verrucomicrobium spinosum (vsTyr) is produced as a pre-pro-enzyme in which a C-terminal extension serves as an inactivation domain. It does not require a caddie protein for copper ion incorporation, which makes it similar to eukaryotic tyrosinases. To gain an understanding of the catalytic machinery and regulation of vsTyr activity, we determined the structure of the catalytically active "core domain" of vsTyr by X-ray crystallography. The analysis showed that vsTyr is an atypical bacterial tyrosinase not only because it is independent of a caddie protein but also because it shows the highest structural (and sequence) similarity to plant-derived members of the type-III copper enzyme family and is more closely related to fungal tyrosinases regarding active site features. By modelling the structure of the pre-pro-enzyme using AlphaFold, we observed that Phe453, located in the C-terminal extension, is appropriately positioned to function as a "gatekeeper" residue. Our findings raise questions concerning the evolutionary origin of vsTyr.
|
|
| 6. |
- Ghosh, Swagatha, et al.
(författare)
-
A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
-
Ingår i: Journal of Applied Crystallography. - 0021-8898. ; 56:Pt 2, s. 449-460
-
Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
|
|
| 7. |
- John, Juliane, et al.
(författare)
-
Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
- 2022
-
Ingår i: eLIFE. - 2050-084X. ; 11
-
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.
|
|
| 8. |
- John, Juliane, et al.
(författare)
-
Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
- 2022
-
Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 11
-
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.
|
|
| 9. |
- Lebrette, Hugo, 1986-, et al.
(författare)
-
Structure of a ribonucleotide reductase R2 protein radical
- 2023
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6666, s. 109-113
-
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.
|
|
| 10. |
- LIZATOVIC, ROBERT, et al.
(författare)
-
A de Novo Designed Coiled-Coil Peptide with a Reversible pH-Induced Oligomerization Switch
- 2016
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 24:6, s. 946-955
-
Tidskriftsartikel (refereegranskat)abstract
- Protein conformational switches have many useful applications but are difficult to design rationally. Here we demonstrate how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold. We describe a de novo designed peptide that was discovered to switch between a parallel symmetric pentamer at pH 8 and a trimer of antiparallel dimers at pH 6. The transition between pentamer and hexamer is caused by changes in the protonation states of glutamatic acid residues with highly upshifted pKa values in both oligomer forms. The drastic conformational change coupled with the narrow pH range makes the peptide sequence an attractive candidate for introduction of pH sensing into other proteins. The results highlight the remarkable ability of simple-α helices to self-assemble into a vast range of structural states.
|
|
