Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Burden and resources in caregivers of people with multiple sclerosis: A qualitative study.

BACKGROUND: Caregivers of people with Multiple Sclerosis are required to provide ongoing assistance especially during the advanced stages of the disease. They have to manage interventions and assume responsibilities which significantly impact both their personal quality of life and family's dynamics. OBJECTIVE: A qualitative phenomenological study was carried out to understand the experience of burden in caregivers and their resources to manage it. The study also explores how healthcare services involved in the Multiple Sclerosis Clinical Pathway respond to the needs of well-being of patients and family members. METHODS: 17 caregivers were involved in focus groups and in semi-structured individual interviews. RESULTS: Fatigue is experienced by all respondents and it starts when physical disabilities increase or when people become aware of them. Many caregivers declare that they refer to intrinsic (love towards their relatives, patience and dedication) or extrinsic (family members, hobbies) resources to cope with the burden of assistance. Patient associations and the Multiple Sclerosis Clinical Pathway play a significant role in supporting caregivers. CONCLUSIONS: Fatigue, loneliness, and isolation are experienced by caregivers and strongly affect their quality of life and health status. The study highlights caregivers' need to reconcile working times with care times, to give more space to self-care and to have moments to share their experiences with someone else. These needs should be at the core of health policies in order to avoid physical and emotional breakdowns which could lead to the rupture of the relational balance on which home care is based.

3-O-Methyltolcapone and Its Lipophilic Analogues Are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinson's disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation that is responsible for the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

Erwinia tasmaniensis levansucrase shows enantiomer selection for (S)-1,2,4-butanetriol.

Levansucrases are biotechnologically interesting fructosyltransferases due to their potential use in the enzymatic or chemo-enzymatic synthesis of glycosides of non-natural substrates relevant to pharmaceutical applications. The structure of Erwinia tasmaniensis levansucrase in complex with (S)-1,2,4-butanetriol and its biochemical characterization suggests the possible application of short aliphatic moieties containing polyols with defined stereocentres in fructosylation biotechnology. The structural information revealed that (S)-1,2,4-butanetriol mimics the natural substrate. The preference of the protein towards a specific 1,2,4-butanetriol enantiomer was assessed using microscale thermophoresis binding assays. Furthermore, the results obtained and the structural comparison of levansucrases and inulosucrases suggest that the fructose binding modes could differ in fructosyltransferases from Gram-positive and Gram-negative bacteria.

The structural and functional characterization of Malus domestica double bond reductase MdDBR provides insights towards the identification of its substrates.

In this study we describe the crystal structures of the apoform, the binary and the ternary complexes of a double bond reductase from Malus domestica L. (MdDBR) and explore a range of potential substrates. The overall fold of MdDBR is similar to that of the medium chain reductase/dehydrogenase/zinc-dependent alcohol dehydrogenase-like family. Structural comparison of MdDBR with Arabidopsis thaliana DBR (AtDBR), Nicotiana tabacum DBR (NtDBR) and Rubus idaeus DBR (RiDBR) allowed the identification of key amino acids involved in cofactor and ligands binding and shed light on how these residues may guide the orientation of the substrates. The enzyme kinetic for the substrate trans-4-phenylbuten-2-one has been analyzed, and MdDBR activity towards a variety of substrates was tested. This enzyme has been reported to be involved in the phenylpropanoid pathway where it would catalyze the NADPH-dependent reduction of the α, ß-unsaturated double bond of carbonyl metabolites. Our study provides new data towards the identification of MdDBR natural substrate and the biosynthetic pathway where it belongs. Furthermore, the originally proposed involvement in dihydrochalcone biosynthesis in apple must be questioned.

Carbohydrate-Active Enzymes: Structure, Activity, and Reaction Products.

Carbohydrate-active enzymes are responsible for both the biosynthesis and breakdown of carbohydrates and glycoconjugates [...].

The Structure of Sucrose-Soaked Levansucrase Crystals from Erwinia tasmaniensis reveals a Binding Pocket for Levanbiose.

Given its potential role in the synthesis of novel prebiotics and applications in the pharmaceutical industry, a strong interest has developed in the enzyme levansucrase (LSC, EC 2.4.1.10). LSC catalyzes both the hydrolysis of sucrose (or sucroselike substrates) and the transfructosylation of a wide range of acceptors. LSC from the Gram-negative bacterium Erwinia tasmaniensis (EtLSC) is an interesting biocatalyst due to its high-yield production of fructooligosaccharides (FOSs). In order to learn more about the process of chain elongation, we obtained the crystal structure of EtLSC in complex with levanbiose (LBS). LBS is an FOS intermediate formed during the synthesis of longer-chain FOSs and levan. Analysis of the LBS binding pocket revealed that its structure was conserved in several related species. The binding pocket discovered in this crystal structure is an ideal target for future mutagenesis studies in order to understand its biological relevance and to engineer LSCs into tailored products.

The Impact of pH on Catalytically Critical Protein Conformational Changes: The Case of the Urease, a Nickel Enzyme.

Urease uses a cluster of two NiII ions to activate a water molecule for urea hydrolysis. The key to this unsurpassed enzyme is a change in the conformation of a flexible structural motif, the mobile flap, which must be able to move from an open to a closed conformation to stabilize the chelating interaction of urea with the NiII cluster. This conformational change brings the imidazole side chain functionality of a critical histidine residue, αHis323, in close proximity to the site that holds the transition state structure of the reaction, facilitating its evolution to the products. Herein, we describe the influence of the solution pH in modulating the conformation of the mobile flap. High-resolution crystal structures of urease inhibited in the presence of N-(n-butyl)phosphoric triamide (NBPTO) at pH 6.5 and pH 7.5 are described and compared to the analogous structure obtained at pH 7.0. The kinetics of urease in the absence and presence of NBPTO are investigated by a calorimetric assay in the pH 6.0-8.0 range. The results indicate that pH modulates the protonation state of αHis323, which was revealed to have pKa =6.6, and consequently the conformation of the mobile flap. Two additional residues (αAsp224 and αArg339) are shown to be key factors for the conformational change. The role of pH in modulating the catalysis of urea hydrolysis is clarified through the molecular and structural details of the interplay between protein conformation and solution acidity in the paradigmatic case of a metalloenzyme.

The Structure of the Elusive Urease-Urea Complex Unveils the Mechanism of a Paradigmatic Nickel-Dependent Enzyme.

Urease, the most efficient enzyme known, contains an essential dinuclear NiII cluster in the active site. It catalyzes the hydrolysis of urea, inducing a rapid pH increase that has negative effects on human health and agriculture. Thus, the control of urease activity is of utmost importance in medical, pharmaceutical, and agro-environmental applications. All known urease inhibitors are either toxic or inefficient. The development of new and efficient chemicals able to inhibit urease relies on the knowledge of all steps of the catalytic mechanism. The short (microseconds) lifetime of the urease-urea complex has hampered the determination of its structure. The present study uses fluoride to substitute the hydroxide acting as the co-substrate in the reaction, preventing the occurrence of the catalytic steps that follow substrate binding. The 1.42 Šcrystal structure of the urease-urea complex, reported here, resolves the enduring debate on the mechanism of this metalloenzyme.

The structure of Erwinia amylovora AvrRpt2 provides insight into protein maturation and induced resistance to fire blight by Malus × robusta 5.

The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2EA) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malus × robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2EA, a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85 Å. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.

The crystal structure of Rv2991 from Mycobacterium tuberculosis: An F420 binding protein with unknown function.

The crystal structure of the conserved hypothetical protein Rv2991 from Mycobacterium tuberculosis has been solved by SAD using seleno-methionine substituted protein. The dimeric biological assembly and the sequence and fold conservation are typical of F420 cofactor binding enzymes. Despite Rv2991 still being of unknown function, sequence and structural comparison with similar proteins enable a role to be proposed for its C-terminal stretch of residues in recognizing and orienting the substrate. In addition, the C-terminus is involved in both protein folding and determining the size of the active site cavity.

A genome-wide analysis of desferrioxamine mediated iron uptake in Erwinia spp. reveals genes exclusive of the Rosaceae infecting strains.

Erwinia amylovora is the etiological agent of fire blight, a devastating disease which is a global threat to commercial apple and pear production. The Erwinia genus includes a wide range of different species belonging to plant pathogens, epiphytes and even opportunistic human pathogens. The aim of the present study is to understand, within the Erwinia genus, the genetic differences between phytopathogenic strains and those strains not reported to be phytopathogenic. The genes related to the hydroxamate siderophores iron uptake have been considered due to their potential druggability. In E. amylovora siderophore-mediated iron acquisition plays a relevant role in the progression of Fire blight. Here we analyzed the taxonomic relations within Erwinia genus and the relevance of the genes related to the siderophore-mediated iron uptake pathway. The results of this study highlight the presence of a well-defined sub-group of Rosaceae infecting species taxonomically and genetically related with a high number of conserved core genes. The analysis of the complete ferrioxamine transport system has led to the identification of two genes exclusively present in the Rosaceae infecting strains.

Comparison of the Levansucrase from the epiphyte Erwinia tasmaniensis vs its homologue from the phytopathogen Erwinia amylovora.

Erwinia tasmaniensis is an epiphytic bacterium related to the plant pathogen Erwinia amylovora, the etiological agent of fire blight. In this study the levansucrase from E. tasmaniensis (EtLsc) has been compared with the homologous enzyme from E. amylovora (EaLsc). We characterized the enzymatic activity and compared the products profile of both enzymes by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detector (HPAEC-PAD). Moreover we determined the crystal structure of EtLsc to understand the structural peculiarity causing the different product profiles of the two homologues. EtLsc exhibits increased efficiency in the production of FOS, resulting in a better catalyst for biotechnological synthesis than EaLsc. Based on our results, we propose that the role of this enzyme in the life cycle of the two bacteria is most likely related to survival, rather than linked to pathogenicity in E. amylovora.

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Šresolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.

A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.

The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

Conservation of Erwinia amylovora pathogenicity-relevant genes among Erwinia genomes.

The Erwinia genus comprises species that are plant pathogens, non-pathogen, epiphytes, and opportunistic human pathogens. Within the genus, Erwinia amylovora ranks among the top 10 plant pathogenic bacteria. It causes the fire blight disease and is a global threat to commercial apple and pear production. We analyzed the presence/absence of the E. amylovora genes reported to be important for pathogenicity towards Rosaceae within various Erwinia strains genomes. This simple bottom-up approach, allowed us to correlate the analyzed genes to pathogenicity, host specificity, and make useful considerations to drive targeted studies.

The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions.

AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.

Characterization and 1.57†Šresolution structure of the key fire blight phosphatase AmsI from Erwinia amylovora.

AmsI is a low-molecular-weight protein tyrosine phosphatase that regulates the production of amylovoran in the Gram-negative bacterium Erwinia amylovora, a specific pathogen of rosaceous plants such as apple, pear and quince. Amylovoran is an exopolysaccharide that is necessary for successful infection. In order to shed light on AmsI, its structure was solved at 1.57 Šresolution at the same pH as its highest measured activity (pH 5.5). In the active site, a water molecule, bridging between the catalytic Arg15 and the reaction-product analogue sulfate, might be representative of the water molecule attacking the phospho-cysteine intermediate in the second step of the reaction mechanism.