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1.
J Biol Inorg Chem ; 29(3): 339-351, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38227199

RESUMO

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.


Assuntos
Domínio Catalítico , Oxirredutases , Especificidade por Substrato , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Pyrobaculum/enzimologia , Pyrobaculum/genética , Modelos Moleculares , Cristalografia por Raios X
2.
Molecules ; 29(2)2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38257271

RESUMO

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.


Assuntos
Deinococcus , Extremófilos , Peróxido de Hidrogênio , Metionina , Racemetionina , Heme , Peroxidases
3.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639208

RESUMO

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Heme/química , Mutação , Peroxidase/química , Peroxidase/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Corantes/química , Corantes/metabolismo , Estabilidade Enzimática , Heme/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Peroxidase/genética , Conformação Proteica
4.
J Struct Biol ; 205(1): 91-102, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30447285

RESUMO

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-ß-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.


Assuntos
Proteínas de Bactérias/química , Ferroproteínas não Heme/química , Synechocystis/química , Ferro , Ligantes , Estrutura Molecular , Domínios Proteicos
5.
Biochim Biophys Acta Bioenerg ; 1858(10): 847-853, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28760394

RESUMO

The ancient metabolism of photoferrotrophy is likely to have played a key role in the biogeochemical cycle of iron on Early Earth leading to the deposition of Banded Iron Formations prior to the emergence of oxygenic photosynthesis. Extant organisms still performing this metabolism provide a convenient window to peer into its molecular mechanisms. Here we report the molecular structure of FoxE, the putative terminal iron oxidase of Rhodobacter ferrooxidans SW2. This protein is organized as a trimer with two hemes and a disulfide bridge per monomer. The distance between hemes, their solvent exposure and the surface electrostatics ensure a controlled electron transfer rate. They also guarantee segregation between electron capture from ferrous iron and electron release to downstream acceptors, which do not favor the precipitation of ferric iron. Combined with the functional characterization of this protein, the structure reveals how iron oxidation can be performed in the periplasmic space of this Gram-negative bacterium at circumneutral pH, while minimizing the risk of mineral precipitation and cell encrustation.


Assuntos
Compostos Ferrosos/química , Ferro/química , Oxirredutases/química , Rhodobacter/química , Sequência de Aminoácidos , Dissulfetos/química , Transporte de Elétrons/fisiologia , Elétrons , Heme/química , Estrutura Molecular , Oxirredução , Oxigênio/química , Fotossíntese/fisiologia
6.
J Biol Inorg Chem ; 21(1): 39-52, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26767750

RESUMO

Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.


Assuntos
Ferro/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Proteínas/metabolismo
7.
J Biol Chem ; 289(41): 28260-70, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25151360

RESUMO

Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity.


Assuntos
Proteínas de Bactérias/química , Entamoeba histolytica/química , Ferro/química , Lisina/química , Oxirredutases/química , Tirosina/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Entamoeba histolytica/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ferro/metabolismo , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismo
8.
Commun Biol ; 7(1): 179, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38351154

RESUMO

The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir's binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir's scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte , Receptores de Superfície Celular/metabolismo
9.
Biochem Biophys Res Commun ; 430(1): 218-24, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23137539

RESUMO

Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD(+)-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD(+) and the RNA binding site does not coincide with the NAD(+) binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD(+)-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.


Assuntos
Proteínas de Bactérias/metabolismo , RNA/metabolismo , Ribonucleases/metabolismo , Sphingomonas/enzimologia , Uridina Difosfato Glucose Desidrogenase/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Cinética , NAD/química , NAD/metabolismo , Dobramento de Proteína , RNA/química , Ribonucleases/química , Uridina Difosfato Glucose Desidrogenase/química
10.
Artigo em Inglês | MEDLINE | ID: mdl-23695576

RESUMO

Rieske proteins and Rieske ferredoxins are ubiquitous electron-transfer metalloproteins that are characterized by a [2Fe-2S] cluster coordinated by pairs of cysteine and histidine residues. The thermoacidophilic archaeon Acidianus ambivalens contains a Rieske ferredoxin termed RFd2, which has an hitherto unknown additional region of 40-44 residues at the C-terminus with a Cx3C motif that introduces a novel disulfide bond within the Rieske fold. RFd2 was crystallized with the aim of determining its three-dimensional structure in order to understand the contribution of this as yet unique disulfide bridge to the function and stability of RFd2. RFd2 crystals were successively improved, increasing their diffraction to 1.9 Šresolution. Molecular replacement did not solve the RFd2 structure, but a highly multiple in-house diffraction data set collected at the Cu Kα edge led to solution of the phase problem.


Assuntos
Acidianus , Dissulfetos/química , Complexo III da Cadeia de Transporte de Elétrons/química , Sequência de Aminoácidos , Cristalografia por Raios X , Complexo III da Cadeia de Transporte de Elétrons/genética , Ferredoxinas/química , Ferredoxinas/genética , Dados de Sequência Molecular , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína
12.
Nat Commun ; 14(1): 7289, 2023 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-37963862

RESUMO

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.


Assuntos
Glicosídeos Cardíacos , Oxirredutases , Oxirredutases/metabolismo , Peróxido de Hidrogênio , Glicosídeos/metabolismo , Glucose/metabolismo , Especificidade por Substrato , Glicosídeo Hidrolases/metabolismo
13.
Nature ; 443(7107): 110-4, 2006 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-16957732

RESUMO

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.


Assuntos
Escherichia coli/enzimologia , Exorribonucleases/química , Exorribonucleases/metabolismo , RNA/química , RNA/metabolismo , Catálise , Escherichia coli/genética , Exorribonucleases/genética , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Eletricidade Estática
14.
ACS Catal ; 12(9): 5022-5035, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-36567772

RESUMO

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

15.
Comput Struct Biotechnol J ; 20: 3899-3910, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35950185

RESUMO

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK a of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications.

16.
J Bacteriol ; 193(15): 3978-87, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21602353

RESUMO

Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderia cepacia/enzimologia , Tirosina/metabolismo , Uridina Difosfato Glucose Desidrogenase/química , Uridina Difosfato Glucose Desidrogenase/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Burkholderia cepacia/química , Burkholderia cepacia/genética , Domínio Catalítico , Estabilidade Enzimática , Ésteres/metabolismo , Cinética , Dados de Sequência Molecular , Tirosina/genética , Uridina Difosfato Glucose Desidrogenase/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-20057075

RESUMO

Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 A resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.


Assuntos
Uridina Difosfato Glucose Desidrogenase/química , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Selenometionina/metabolismo , Sphingomonas , Uridina Difosfato Glucose Desidrogenase/isolamento & purificação
18.
Artigo em Inglês | MEDLINE | ID: mdl-20208157

RESUMO

Bacteria of the Burkholderia cepacia complex (Bcc) have emerged as important opportunistic pathogens, establishing lung infections in immunocompromised or cystic fibrosis patients. Bcc uses polysaccharide-biofilm production in order to evade the host immune response. The biofilm precursor UDP-glucuronic acid is produced by a twofold NAD(+)-dependent oxidation of UDP-glucose. In B. cepacia IST408 this enzymatic reaction is performed by the UDP-glucose dehydrogenase BceC, a 470-residue enzyme, the production and crystallization of which are described here. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and contained four molecules in the asymmetric unit. Their crystallographic analysis at 2.09 A resolution and a molecular-replacement study are reported.


Assuntos
Burkholderia cepacia/enzimologia , Uridina Difosfato Glucose Desidrogenase/química , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Expressão Gênica , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/isolamento & purificação
19.
Zootaxa ; 4638(3): zootaxa.4638.3.8, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31712472

RESUMO

The third instar of Macraspis clavata (Olivier, 1789), collected in Rio Grande do Sul, Brazil, is described. Illustrations, comments on the natural history, and a key to third instar of Macraspis are also added.


Assuntos
Besouros , Trematódeos , Animais , Brasil
20.
Free Radic Biol Med ; 140: 36-60, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30735841

RESUMO

Microbial anaerobes are exposed in the natural environment and in their hosts, even if transiently, to fluctuating concentrations of oxygen and its derived reactive species, which pose a considerable threat to their anoxygenic lifestyle. To counteract these stressful conditions, they contain a multifaceted array of detoxifying systems that, in conjugation with cellular repairing mechanisms and in close crosstalk with metal homeostasis, allow them to survive in the presence of O2 and reactive oxygen species. Some of these systems are shared with aerobes, but two families of enzymes emerged more recently that, although not restricted to anaerobes, are predominant in anaerobic microbes. These are the iron-containing superoxide reductases, and the flavodiiron proteins, endowed with O2 and/or NO reductase activities, which are the subject of this Review. A detailed account of their physicochemical, physiological and molecular mechanisms will be presented, highlighting their unique properties in allowing survival of anaerobes in oxidative stress conditions, and comparing their properties with the most well-known detoxifying systems.


Assuntos
Ferro/metabolismo , Estresse Oxidativo , Oxirredutases/metabolismo , Proteínas/metabolismo , Anaerobiose/genética , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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