Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.

D-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography-small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.

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.

Unravelling Novel SCN5A Mutations Linked to Brugada Syndrome: Functional, Structural, and Genetic Insights.

Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia causing potentially fatal ventricular tachycardia or fibrillation, mainly occurring during rest or sleep in young individuals without heart structural issues. It increases the risk of sudden cardiac death, and its characteristic feature is an abnormal ST segment elevation on the ECG. While BrS has diverse genetic origins, a subset of cases can be conducted to mutations in the SCN5A gene, which encodes for the Nav1.5 sodium channel. Our study focused on three novel SCN5A mutations (p.A344S, p.N347K, and p.D349N) found in unrelated BrS families. Using patch clamp experiments, we found that these mutations disrupted sodium currents: p.A344S reduced current density, while p.N347K and p.D349N completely abolished it, leading to altered voltage dependence and inactivation kinetics when co-expressed with normal channels. We also explored the effects of mexiletine treatment, which can modulate ion channel function. Interestingly, the p.N347K and p.D349N mutations responded well to the treatment, rescuing the current density, while p.A344S showed a limited response. Structural analysis revealed these mutations were positioned in key regions of the channel, impacting its stability and function. This research deepens our understanding of BrS by uncovering the complex relationship between genetic mutations, ion channel behavior, and potential therapeutic interventions.

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.

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.

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.

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).

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.

Comparison of helical scan and standard rotation methods in single-crystal X-ray data collection strategies.

X-ray radiation in macromolecular crystallography can chemically alter the biological material and deteriorate the integrity of the crystal lattice with concomitant loss of resolution. Typical alterations include decarboxylation of glutamic and aspartic residues, breaking of disulfide bonds and the reduction of metal centres. Helical scans add a small translation to the crystal in the rotation method, so that for every image the crystal is shifted to expose a fresh part. On beamline PROXIMA 2A at Synchrotron SOLEIL, this procedure has been tested with various parameters in an attempt to understand how to mitigate the effects of radiation damage. Here, the strategies used and the crystallographic metrics for various scenarios are reported. Among these, the loss of bromine from bromophenyl moieties appears to be a useful monitor of radiation damage as the carbon-bromine bond is very sensitive to X-ray irradiation. Two cases are focused on where helical scans are shown to be superior in obtaining meaningful data compared with conventional methods. In one case the initial resolution of the crystal is extended over time, and in the second case the anomalous signal is preserved to provide greater effective multiplicity and easier phasing.

A new crystal form of human transthyretin obtained with a curcumin derived ligand.

Transthyretin (TTR), a 54kDa homotetrameric protein that transports thyroxine (T4), has been associated with clinical cases of TTR amyloidosis for its tendency to aggregate to form fibrils. Many ligands with a potential to inhibit fibril formation have been studied by X-ray crystallography in complex with TTR. Unfortunately, the ligand is often found in ambiguous electron density that is difficult to interpret. The ligand validation statistics suggest over-interpretation, even for the most active compounds like diflunisal. The primary technical reason is its position on a crystallographic 2-fold axis in the most common crystal form. Further investigations with the use of polyethylene glycol (PEG) to crystallize TTR complexes have resulted in a new trigonal polymorph with two tetramers in the asymmetric unit. The ligand used to obtain this new polymorph, 4-hydroxychalcone, is related to curcumin. Here we evaluate this crystal form to understand the contribution it may bring to the study of TTR ligands complexes, which are often asymmetric.