The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

Reviewing a plethora of oxidative-type reactions catalyzed by whole cells of Streptomyces species.

Selective oxidation reactions represent a challenging task for conventional organic chemistry. Whole-cell biocatalysis provides a very convenient, easy to apply method to carry out different selective oxidation reactions including chemo-, regio-, and enantio-selective reactions. Streptomyces species are important biocatalysts as they can catalyze these selective reactions very efficiently owing to the wide diversity of enzymes and enzymatic cascades in their cell niche. In this review, we present and analyze most of the examples reported to date of oxidative reactions catalyzed by Streptomyces species as whole-cell biocatalysts. We discuss 33 different Streptomyces species and strains and the role they play in different oxidative reactions over the past five decades. The oxidative reactions have been classified into seven categories that include: hydroxylation of steroids/non-steroids, asymmetric sulfoxidations, oxidation of aldehydes, multi-step oxidations, oxidative cleavage, and N-oxidations. The role played by Streptomyces species as recombinant hosts catalyzing bio-oxidations has also been highlighted.

Solid State NMR a Powerful Technique for Investigating Sustainable/Renewable Cellulose-Based Materials.

Solid state nuclear magnetic resonance (ssNMR) is a powerful and attractive characterization method for obtaining insights into the chemical structure and dynamics of a wide range of materials. Current interest in cellulose-based materials, as sustainable and renewable natural polymer products, requires deep investigation and analysis of the chemical structure, molecular packing, end chain motion, functional modification, and solvent-matrix interactions, which strongly dictate the final product properties and tailor their end applications. In comparison to other spectroscopic techniques, on an atomic level, ssNMR is considered more advanced, especially in the structural analysis of cellulose-based materials; however, due to a dearth in the availability of a broad range of pulse sequences, and time consuming experiments, its capabilities are underestimated. This critical review article presents the comprehensive and up-to-date work done using ssNMR, including the most advanced NMR strategies used to overcome and resolve the structural difficulties present in different types of cellulose-based materials.

Rediscovering bacterial exopolysaccharides of terrestrial and marine origins: novel insights on their distribution, biosynthesis, biotechnological production, and future perspectives.

Bacteria exist in colonies as aggregates or associated with surfaces forming biofilms rather than planktonic cells. Living in such a unique manner is always mediated via a matrix of extracellular polymeric substances, which are composed mainly of polysaccharides or specifically exopolysaccharides (EPS). Biofilm formation and hence EPS production are affected by biotic and abiotic factors inducing/inhibiting several involved genes and other molecules. In addition, various aspects of bacterial EPS regarding: physiological functions, molecular weight, and chemical composition were demonstrated. Recent investigations have revealed a wide spectrum of EPS chemical and physicochemical properties showing promising applications in different industrial sectors. For instance, lactic acid bacteria (LAB)- and marine-derived EPS exhibit: immunomodulatory, antioxidant, antitumor, bioremediation of heavy metals, as well as thickening and viscosity modifiers in the food industry. However, bacterial EPS have not yet been commercially implemented, in contrast to plant-derived analogues. The current review aims to rediscover the EPS structural and biosynthetic features derived from marine and terrestrial bacteria, and applications as well.

Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.

Enantioselective sulfoxidation using Streptomyces glaucescens GLA.0.

Asymmetric oxidation of prochiral sulfides is a direct means for production of enantiopure sulfoxides which are important in organic synthesis and the pharmaceutical industry. In the present study, Streptomyces glaucescens GLA.0 was employed for stereoselective oxidation of prochiral sulfides. Growing cells selectively catalyzed the oxidation of phenyl methyl sulfide to the corresponding sulfoxide. Only very little overoxidation was observed, resulting in minor amounts of the unwanted sulfone. Addition of isopropyl alcohol as a co-solvent, time of substrate addition and composition of the reaction media resulted in enhanced phenyl methyl sulfide biotransformation. The concentration of the undesired by-product (sulfone) was as low as 4% through the reaction course under optimal reaction conditions. The results show that S. glaucescens GLA.0 is a promising whole-cell biocatalyst for preparing highly enantiopure (R)-phenyl methyl sulfoxide in high yield (90%) with an enantiomeric excess (ee) exceeding 99%.

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis.

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Bacterial enzymes involved in lignin degradation.

Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)processing of lignocellulosic feedstocks, more effective degradation methods of lignin are in demand. Nature has found ways to fully degrade lignin through the production of dedicated ligninolytic enzyme systems. While such enzymes have been well thoroughly studied for ligninolytic fungi, only in recent years biochemical studies on bacterial enzymes capable of lignin modification have intensified. This has revealed several types of enzymes available to bacteria that enable them to act on lignin. Two major classes of bacterial lignin-modifying enzymes are DyP-type peroxidases and laccases. Yet, recently also several other bacterial enzymes have been discovered that seem to play a role in lignin modifications. In the present review, we provide an overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignin-derived products.