Evolution of foot-and-mouth disease virus intra-sample sequence diversity during serial transmission in bovine hosts

RNA virus populations within samples are highly heterogeneous, containing a large number of minority sequence variants which can potentially be transmitted to other susceptible hosts. Consequently, consensus genome sequences provide an incomplete picture of the within- and between-host viral evolutionary dynamics during transmission. Foot-and-mouth disease virus (FMDV) is an RNA virus that can spread from primary sites of replication, via the systemic circulation, to found distinct sites of local infection at epithelial surfaces. Viral evolution in these different tissues occurs independently, each of them potentially providing a source of virus to seed subsequent transmission events. This study employed the Illumina Genome Analyzer platform to sequence 18 FMDV samples collected from a chain of sequentially infected cattle. These data generated snap-shots of the evolving viral population structures within different animals and tissues. Analyses of the mutation spectra revealed polymorphisms at frequencies >0.5% at between 21 and 146 sites across the genome for these samples, while 13 sites acquired mutations in excess of consensus frequency (50%). Analysis of polymorphism frequency revealed that a number of minority variants were transmitted during host-to-host infection events, while the size of the intra-host founder populations appeared to be smaller. These data indicate that viral population complexity is influenced by small intra-host bottlenecks and relatively large inter-host bottlenecks. The dynamics of minority variants are consistent with the actions of genetic drift rather than strong selection. These results provide novel insights into the evolution of FMDV that can be applied to reconstruct both intra- and inter-host transmission routes

Synthesis of γ-glutamyl derivatives of sulfur-containing amino acids in a multigram scale via a two-step, one-pot procedure

\u3b3-Glutamyl derivatives of sulfur amino acids have been prepared in multigram scale starting from readily available starting materials. The synthesis comprises two one-pot operations, both consisting of two reactions. In the first operation, N-phtaloyl-l-glutamic acid anhydride is obtained from l-glutamic acid and phtalic anhydride. In the second one, N-phtaloyl-l-glutamic acid anhydride is used to acylate amino acids and the N-phtaloyl protecting group is removed. The described approach offers a viable entry to \u3b3-glutamyl derivatives of sulfur-containing amino acids with flavor-enhancer and nutraceutical properties

Enzymatic synthesis of γ-glutamyl derivatives catalyzed by a new mutant γ-glutamyltransferase with improved transpeptidase activity

Despite their potential applicative interest as biologically active compounds and as flavor enhancers, \u3b3-glutamyl derivatives are commercially underexploited compounds. This is mainly due to the difficulties connected with their supply at a reasonable cost. As a consequence, enzymatic approaches to their preparation, based on the use of \u3b3-glutamyltransferases (GGTs), have been proposed1 to circumvent both the low-yielding extractive procedures from natural sources and the troublesome chemical synthesis, rendered uneconomical by the need of protection and deprotection steps. GGTs catalyze the transfer of a \u3b3-glutamyl moiety from a donor substrate (e.g. glutathione) to the primary amino group of an acceptor compound in a so-called transpeptidation reaction, through the formation of a \u3b3-glutamyl-enzyme intermediate. However, also the use of GGTs as biocatalysts is not free from drawbacks. In addition to the transpeptidase activity, GGTs show a non-negligible hydrolase activity towards both the donor substrate and the newly formed transpeptidation product, affording irreversibly glutamic acid.2 In our ongoing studies on bacterial GGTs, we found that the presence of the lid loop \u2013 a short amino acids sequence covering the active site in most of the known GGTs \u2013 not only affects substrate selection, but also modulates hydrolase/transpeptidase activities.3 Within the TailGluTran Project,4 aimed at the development of mutant GGTs with improved transpeptidase activity, is currently under investigation a mutant enzyme obtained by inserting the sequence of the lid loop on the structure of a GGT naturally lacking it. The mutant enzyme shows promising high transpeptidase activity with respect to wild type counterparts and represents a starting point for further modifications in the search of a suitable biocatalyst intended for preparative purposes

Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection

\u3b3-Glutamyltransferase (GGT) catalyzes the transfer of the \u3b3-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a \u3b3-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-\u3b3-glutamic acid (\u3b3-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-\u3b3-glutamylglutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity

A mutant γ-glutamyltransferase with improved transpeptidase activity

Despite their potential applicative interest as biologically active compounds and as flavor enhancers, \u3b3-glutamyl derivatives are commercially underexploited compounds. This is mainly due to the difficulties connected with their supply at a reasonable cost. As a consequence, enzymatic approaches to their preparation, based on the use of \u3b3-glutamyltransferases (GGTs), have been proposed1 to circumvent both the low-yielding extractive procedures from natural sources and the troublesome chemical synthesis, rendered uneconomical by the need of protection and deprotection steps. GGTs catalyze the transfer of a \u3b3-glutamyl moiety from a donor substrate (e.g. glutathione or glutamine) to the primary amino group of an acceptor compound in a so-called transpeptidation reaction through the formation of a \u3b3-glutamyl-enzyme intermediate. However, also the use of GGTs as biocatalysts is not free from drawbacks. In addition to the transpeptidase activity, GGTs show a non-negligible hydrolase activity towards both the donor substrate and the newly formed transpeptidation product, affording irreversibly glutamic acid.2 In our ongoing studies on bacterial GGTs, we found that the presence of the lid loop \u2013 a short amino acids sequence covering the active site in most of the known GGTs \u2013 not only affects substrate selection, but also modulates hydrolase/transpeptidase activities.3 Within the TailGluTran Project,4 aimed at the development of mutant GGTs with improved transpeptidase activity, is currently under investigation a mutant enzyme obtained by inserting the sequence of the lid loop on the structure of a GGT naturally lacking it. The mutant enzyme shows promising high transpeptidase activity with respect to wild type counterparts and represents a starting point for further modifications in the search of a suitable biocatalyst intended for preparative purposes

Chemistry of α-mangostin : studies on the semisynthesis of minor xanthones from Garcinia mangostana

\u3b1-Mangostin is the major prenylated xanthone from Garcinia mangostana and it has been used also in recent times as starting material for the semisynthetic preparation of various biologically active derivatives. Its structure is characterised by the presence of few functional groups amenable to chemical manipulations, but present in the molecule in multiple instances (three phenolic hydroxyl groups, two prenyl chains and two unsubstituted aromatic carbons). This study represents a first approach to the systematic investigation of the reactivity of \u3b1-mangostin and describes the semisynthesis of some minor xanthones isolated from G. mangostana

Biocatalysis for biomass valorization: peptides and fatty acids from rice bran

Waste upgrading practises have attracted a significant attention in recent years with the aim of managing agrofood by-products in a gainful and sustainable way. We describe here how biocatalysis can assist rice bran valorization, according to the biorefinery concept. [1] Rice is the staple food for over half the world's population. Rice milling generates a massive amount of waste, namely rice bran (70 kg/ton of rice) and rice husk (200 kg/ton of rice). Rice bran (RB), containing fibers (7-11%), proteins (10-16%), lipids (15-22%), carbohydrates (34-52%), micronutrients, represents a second-generation biomass. [2] Rice bran proteins (RBP) have a high nutritional value and optimal digestibility and are gluten-free, hypoallergenic and rich in essential amino acids. However, the first hurdle to be overcome for RBP production and large scale application is their extraction. Structural complexity, poor solubility, and strong aggregation make RBP hardly available. The sequential treatment of RB with carbohydrases and proteases was used to prepare mixtures of water-soluble peptides (RBPHs, RBP Hydrolysates) to be tested as antibacterial, antioxidant and anticholesterol agents, as well as flavour enhancers. [3] Interestingly, sensory analysis revealed that the obtained RBPHs exert only sweet and umami taste. Rice bran oil (RBO) is one of the most underutilized agricultural commodities. We investigated the use of RBO as a feedstock for the production of FFA-derived chemicals (e.g. sugar fatty acid esters). [4] To this aim, RBO was submitted to a preparative lipase-catalyzed hydrolysis to obtain pure FFA. [5] The high acidity of RBO, so far considered as a bottleneck in the exploitation of RBO (i.e. biodiesel production) was here turned into an advantage, making available FFA mixtures as synthetic precursors for high added value products

The structure of PghL hydrolase bound to its substrate poly-γ-glutamate

The identification of new strategies to fight bacterial infections in view of the spread of multiple resistance to antibiotics has become mandatory. It has been demonstrated that several bacteria develop poly-?-glutamic acid (?-PGA) capsules as a protection from external insults and/or host defence systems. Among the pathogens that shield themselves in these capsules are Bacillus\ua0anthracis, Francisella\ua0tularensis and several Staphylococcus strains. These are important pathogens with a profound influence on human health. The recently characterised ?-PGA hydrolases, which can dismantle the ?-PGA-capsules, are an attractive new direction that can offer real hope for the development of alternatives to antibiotics, particularly in cases of multidrug resistant bacteria. We have characterised in detail the cleaving mechanism and stereospecificity of the enzyme PghL (previously named YndL) from Bacillus\ua0subtilis encoded by a gene of phagic origin and dramatically efficient in degrading the long polymeric chains of ?-PGA. We used X-ray crystallography to solve the three-dimensional structures of the enzyme in its zinc-free, zinc-bound and complexed forms. The protein crystallised with a ?-PGA hexapeptide substrate and thus reveals details of the interaction which could explain the stereospecificity observed and give hints on the catalytic mechanism of this class of hydrolytic enzymes

Batch and Flow Synthesis of Nucleosides by Enzymatic Transglycosylation

Enzymatic methods for the preparation of high-value products have clearly shown their potential in many areas, including nucleic acid chemistry. Enzymes of nucleic acid metabolism such as nucleoside phosphorylases (NPs, EC 2.4.2) can be conveniently used as biocatalysts in the synthesis of nucleoside analogues. These enzymes catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic phosphate (Pi) to generate the nucleobase and \u3b1-D-(deoxy)ribose-1-phosphate (phosphorolysis). If a second nucleobase is added to the reaction medium, the formation of a new nucleoside can result (transglycosylation). Because of its broad substrate specificity [1,2], a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) was exploited to catalyze the \u201cone-pot, one-enzyme\u201d transglycosylation of 7-methylguanosine iodide with a series of 6-substituted purines, resulting in a moderate to high conversion (18-65%) of the bases into a 22-compound library of 6-substituted purine ribonucleosides [2]. Successively, AhPNP was covalently immobilized [3,4] in a pre-packed column containing aminopropyl silica particles. The resulting AhPNP-IMER (Immobilized Enzyme Reactor) was coupled on-line to a HPLC apparatus containing a semi-preparative chromatographic column. In such a system, \u201cone-enzyme\u201d transglycosylation and product purification were run in a single platform, affording a set of 6-modified purine ribonucleosides at a mg scale [4]. Using this \u201cflow-based\u201d approach, the synthesis of adenine nucleosides through a \u201ctwo-enzyme\u201d transglycosylation was carried out by connecting the AhPNP-IMER to uridine phosphorylase from Clostridium perfringens, immobilized on a silica monolithic column (CpUP-IMER)

Synthesis of ribavirin, tecadenoson, and cladribine by enzymatic transglycosylation

Despite the impressive progress in nucleoside chem. to date, the synthesis of nucleoside analogs is still a challenge. Chemoenzymic synthesis has been proven to overcome most of the constraints of conventional nucleoside chem. A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) has been used herein to catalyze the synthesis of Ribavirin, Tecadenoson, and Cladribine, by a "one-pot, one-enzyme" transglycosylation, which is the transfer of the carbohydrate moiety from a nucleoside donor to a heterocyclic base. As the sugar donor, 7-methylguanosine iodide and its 2'-deoxy counterpart were synthesized and incubated either with the "purine-like" base or the modified purine of the three selected APIs. Good conversions (49-67%) were achieved in all cases under screening conditions. Following this synthetic scheme, 7-methylguanine arabinoside iodide was also prepd. with the purpose to synthesize the antiviral Vidarabine by a novel approach. However, in this case, neither the phosphorolysis of the sugar donor, nor the transglycosylation reaction were obsd. This study was enlarged to two other ribonucleosides structurally related to Ribavirin and Tecadenoson, namely, Acadesine, or AICAR, and 2-chloro-N6-cyclopentyladenosine, or CCPA. Only the formation of CCPA was obsd. (52%). This study paves the way for the development of a new synthesis of the target APIs at a preparative scale. Furthermore, the screening herein reported contributes to the collection of new data about the specific substrate requirements of AhPNP. [on SciFinder(R)