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1.
Proc Natl Acad Sci U S A ; 116(42): 20984-20990, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31570616

RESUMEN

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.


Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fotosíntesis/efectos de la radiación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cianobacterias/química , Cianobacterias/genética , Cianobacterias/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Luz , NADP/química , NADP/metabolismo , Oxidación-Reducción/efectos de la radiación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Thermosynechococcus
2.
Proc Natl Acad Sci U S A ; 115(33): E7824-E7833, 2018 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-30061392

RESUMEN

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Complejo de Proteína del Fotosistema II/biosíntesis , Proteínas Bacterianas/genética , Cianobacterias/genética , Complejo de Proteína del Fotosistema I/biosíntesis , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema II/genética
3.
J Biol Chem ; 294(24): 9367-9376, 2019 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-31043481

RESUMEN

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.


Asunto(s)
Azotobacter vinelandii/enzimología , Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Nitrógeno/metabolismo , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Fijación del Nitrógeno , Oxidación-Reducción , Oxidorreductasas/química , Conformación Proteica
4.
Proc Natl Acad Sci U S A ; 113(37): 10346-51, 2016 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-27573845

RESUMEN

The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops.


Asunto(s)
Péptidos/química , Conformación Proteica , Proteínas/química , Secuencias Repetitivas de Aminoácido/genética , Secuencia de Aminoácidos/genética , Cristalografía por Rayos X , Péptidos/genética , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas/genética
5.
Mol Biol Evol ; 32(5): 1310-28, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25657330

RESUMEN

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages toward the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.


Asunto(s)
Cianobacterias/genética , Evolución Molecular , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Cianobacterias/metabolismo , Luz , Oxidación-Reducción , Oxígeno/metabolismo , Agua/metabolismo
6.
Plant Cell Physiol ; 57(1): 82-94, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26574578

RESUMEN

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.


Asunto(s)
Chlamydomonas reinhardtii/enzimología , Lactato Deshidrogenasas/metabolismo , Piruvatos/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/genética , Cloroplastos/enzimología , Cloroplastos/genética , Fermentación , Lactato Deshidrogenasas/genética , Modelos Biológicos , Modelos Estructurales , Oxidación-Reducción , Piruvato Descarboxilasa/genética , Piruvato Descarboxilasa/metabolismo
7.
Proc Natl Acad Sci U S A ; 110(4): 1327-32, 2013 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-23297211

RESUMEN

Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Fenómenos Biofísicos , Proteínas Portadoras/genética , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/metabolismo
8.
Biochim Biophys Acta ; 1837(10): 1821-34, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25107631

RESUMEN

The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6(•+), in the modified BFR reconstituted with both ZnCe6 and Mn(II), is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound Mn(II) is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6(•+) and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis.


Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Ferritinas/metabolismo , Modelos Moleculares , Fotosíntesis , Ingeniería de Proteínas , Tirosina/química , Proteínas Bacterianas/química , Secuencia de Bases , Grupo Citocromo b/química , Cartilla de ADN , Espectroscopía de Resonancia por Spin del Electrón , Ferritinas/química , Oxidación-Reducción , Fotoquímica , Reacción en Cadena de la Polimerasa
9.
J Biol Chem ; 288(11): 7803-7814, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23349464

RESUMEN

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.


Asunto(s)
Proteínas Portadoras/química , Proteínas Cullin/química , Ubiquitina-Proteína Ligasas/química , Sitios de Unión , Calorimetría/métodos , Cristalografía por Rayos X/métodos , Dimerización , Humanos , Modelos Moleculares , Conformación Molecular , Filogenia , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina-Proteína Ligasas/metabolismo
10.
Photosynth Res ; 122(1): 57-67, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24838684

RESUMEN

The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the Mn4CaO5 oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ.


Asunto(s)
Cianobacterias/química , Modelos Moleculares , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Cianobacterias/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Peroxirredoxinas/metabolismo , Complejo de Proteína del Fotosistema II/aislamiento & purificación , Complejo de Proteína del Fotosistema II/metabolismo , Estructura Secundaria de Proteína , Alineación de Secuencia , Tilacoides/metabolismo
11.
Biochimie ; 219: 12-20, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37952891

RESUMEN

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and this is further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.


Asunto(s)
Chloroflexus , Malonil Coenzima A , Oxidorreductasas , Cinética , Oxidorreductasas/metabolismo , Malonil Coenzima A/metabolismo , Malonatos
12.
Acta Crystallogr D Struct Biol ; 80(Pt 8): 599-604, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38984904

RESUMEN

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.


Asunto(s)
Azotobacter vinelandii , Proteínas Bacterianas , Modelos Moleculares , Azotobacter vinelandii/química , Cristalografía por Rayos X , Proteínas Bacterianas/química , Conformación Proteica , Dominios Proteicos , Ferredoxinas/química , Multimerización de Proteína , Proteínas Hierro-Azufre/química
14.
J Comput Chem ; 34(22): 1881-9, 2013 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-23703289

RESUMEN

Coarse-grained protein structure models offer increased efficiency in structural modeling, but these must be coupled with fast and accurate methods to revert to a full-atom structure. Here, we present a novel algorithm to reconstruct mainchain models from C traces. This has been parameterized by fitting Gaussian mixture models (GMMs) to short backbone fragments centered on idealized peptide bonds. The method we have developed is statistically significantly more accurate than several competing methods, both in terms of RMSD values and dihedral angle differences. The method produced Ramachandran dihedral angle distributions that are closer to that observed in real proteins and better Phaser molecular replacement log-likelihood gains. Amino acid residue sidechain reconstruction accuracy using SCWRL4 was found to be statistically significantly correlated to backbone reconstruction accuracy. Finally, the PD2 method was found to produce significantly lower energy full-atom models using Rosetta which has implications for multiscale protein modeling using coarse-grained models. A webserver and C++ source code is freely available for noncommercial use from: http://www.sbg.bio.ic.ac.uk/phyre2/PD2_ca2main/.


Asunto(s)
Algoritmos , Carbono/química , Simulación de Dinámica Molecular , Proteínas/química , Programas Informáticos , Conformación Proteica
15.
Photosynth Res ; 117(1-3): 375-83, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24126792

RESUMEN

Members of the Psb28 family of proteins are accessory factors implicated in the assembly and repair of the photosystem II complex. We present here the crystal structure of the Psb28 protein (Tlr0493) found in the thermophilic cyanobacterium Thermosynechococcus elongatus at a resolution of 2.3 Å. Overall the crystal structure of the Psb28 monomer is similar to the solution structures of C-terminally His-tagged Psb28-1 from Synechocystis sp. PCC 6803 obtained previously by nuclear magnetic resonance spectroscopy. One new aspect is that Escherichia coli-expressed T. elongatus Psb28 is able to form dimers in solution and packs as a dimer of dimers in the crystal. Analysis of wild type and mutant strains of Synechocystis 6803 by blue native-polyacrylamide gel electrophoresis suggests that Psb28-1, the closest homologue to T. elongatus Psb28 in this organism, also exists as an oligomer in vivo, most likely a dimer. In line with the prediction based on the crystal structure of T. elongatus Psb28, the addition of a 3× Flag-tag to the C-terminus of Synechocystis 6803 Psb28-1 interferes with the accumulation of the Psb28-1 oligomer in vivo. In contrast, the more distantly related Psb28-2 protein found in Synechocystis 6803 lacks the residues that stabilize dimer formation in the T. elongatus Psb28 crystal and is detected as a monomer in vivo. Overall our data suggest that the dimer interface in the Psb28 crystal might be physiologically relevant.


Asunto(s)
Proteínas Bacterianas/química , Complejo de Proteína del Fotosistema II/química , Synechococcus/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Soluciones , Homología Estructural de Proteína
16.
Biodes Res ; 5: 0005, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37849466

RESUMEN

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases-enzymes capable of converting atmospheric nitrogen N2 to NH3 in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.

17.
Nat Commun ; 14(1): 4681, 2023 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-37542031

RESUMEN

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.


Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Complejo de Proteína del Fotosistema II/metabolismo , Manganeso/metabolismo , Oxígeno/metabolismo , Microscopía por Crioelectrón , Cianobacterias/metabolismo
18.
Nat Rev Chem ; 7(3): 184-201, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37117902

RESUMEN

The Mo/Fe nitrogenase enzyme is unique in its ability to efficiently reduce dinitrogen to ammonia at atmospheric pressures and room temperature. Should an artificial electrolytic device achieve the same feat, it would revolutionize fertilizer production and even provide an energy-dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on solid electrodes, homogeneous catalysts and nitrogenases. Specific emphasis is placed on systems for which there is unequivocal evidence that dinitrogen reduction has taken place. By establishing the cross-cutting themes and synergies between these systems, we identify viable avenues for future research.

19.
Photosynth Res ; 110(3): 177-84, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22187288

RESUMEN

Photosystem II is the oxygen-evolving enzyme of photosynthesis. It is a membrane-bound protein-pigment complex. The oxygen is produced at the oxygen-evolving centre (OEC), a Mn(4)CaO(5) metallocluster, which is largely ligated by amino acids of the D1 protein. The OEC-ligating residues are invariant between most cyanobacteria and higher plants. In this study, a new class of cyanobacterial D1 proteins has been identified in which the OEC metal-ligating residues are very different to the consensus. This new class of 'rogue' D1 proteins is associated with diazotrophic cyanobacteria. Their function, activity and origins are discussed.


Asunto(s)
Cianobacterias/metabolismo , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Consenso , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema II/genética , Filogenia , Alineación de Secuencia
20.
Photosynth Res ; 110(3): 169-75, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22193820

RESUMEN

The biogenesis and oxygen-evolving activity of cyanobacterial Photosystem II (PSII) is dependent on a number of accessory proteins not found in the crystallised dimeric complex. These include Psb27, a small lipoprotein attached to the lumenal side of PSII, which has been assigned a role in regulating the assembly of the Mn(4)Ca cluster catalysing water oxidation. To gain a better understanding of Psb27, we have determined in this study the crystal structure of the soluble domain of Psb27 from Thermosynechococcus elongatus to a resolution of 1.6 Å. The structure is a four-helix bundle, similar to the recently published solution structures of Psb27 from Synechocystis PCC 6803 obtained by nuclear magnetic resonance (NMR) spectroscopy. Importantly, the crystal structure presented here helps us resolve the differences between the NMR-derived structural models. Potential binding sites for Psb27 within PSII are discussed in light of recent biochemical data in the literature.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Secuencia de Aminoácidos , Biología Computacional , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de Proteína del Fotosistema II/química , Unión Proteica , Alineación de Secuencia , Homología Estructural de Proteína
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