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

Euclidean Gibbs states of interacting quantum anharmonic oscillators

A rigorous description of the equilibrium thermodynamic properties of an infinite system of interacting $\nu$-dimensional quantum anharmonic oscillators is given. The oscillators are indexed by the elements of a countable set $\mathbb{L}\subset \mathbb{R}^d$, possibly irregular; the anharmonic potentials vary from site to site. The description is based on the representation of the Gibbs states in terms of path measures -- the so called Euclidean Gibbs measures. It is proven that: (a) the set of such measures $\mathcal{G}^{\rm t}$ is non-void and compact; (b) every $\mu \in \mathcal{G}^{\rm t}$ obeys an exponential integrability estimate, the same for the whole set $\mathcal{G}^{\rm t}$; (c) every $\mu \in \mathcal{G}^{\rm t}$ has a Lebowitz-Presutti type support; (d) $\mathcal{G}^{\rm t}$ is a singleton at high temperatures. In the case of attractive interaction and $\nu=1$ we prove that $|\mathcal{G}^{\rm t}|>1$ at low temperatures. The uniqueness of Gibbs measures due to quantum effects and at a nonzero external field are also proven in this case. Thereby, a qualitative theory of phase transitions and quantum effects, which interprets most important experimental data known for the corresponding physical objects, is developed. The mathematical result of the paper is a complete description of the set $\mathcal{G}^{\rm t}$, which refines and extends the results known for models of this type.Comment: 60 page

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

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

Neutron capture measurement at the n TOF facility of the 204Tl and 205Tl s-process branching points

Neutron capture cross sections are one of the fundamental nuclear data in the study of the s (slow) process of nucleosynthesis. More interestingly, the competition between the capture and the decay rates in some unstable nuclei determines the local isotopic abundance pattern. Since decay rates are often sensible to temperature and electron density, the study of the nuclear properties of these nuclei can provide valuable constraints to the physical magnitudes of the nucleosynthesis stellar environment. Here we report on the capture cross section measurement of two thallium isotopes, 204Tl and 205Tl performed by the time-of-flight technique at the n TOF facility at CERN. At some particular stellar s-process environments, the decay of both nuclei is strongly enhanced, and determines decisively the abundance of two s-only isotopes of lead, 204Pb and 205Pb. The latter, as a long-lived radioactive nucleus, has potential use as a chronometer of the last s-process events that contributed to final solar isotopic abundances

COMODO: an adaptive coclustering strategy to identify conserved coexpression modules between organisms

Increasingly large-scale expression compendia for different species are becoming available. By exploiting the modularity of the coexpression network, these compendia can be used to identify biological processes for which the expression behavior is conserved over different species. However, comparing module networks across species is not trivial. The definition of a biologically meaningful module is not a fixed one and changing the distance threshold that defines the degree of coexpression gives rise to different modules. As a result when comparing modules across species, many different partially overlapping conserved module pairs across species exist and deciding which pair is most relevant is hard. Therefore, we developed a method referred to as conserved modules across organisms (COMODO) that uses an objective selection criterium to identify conserved expression modules between two species. The method uses as input microarray data and a gene homology map and provides as output pairs of conserved modules and searches for the pair of modules for which the number of sharing homologs is statistically most significant relative to the size of the linked modules. To demonstrate its principle, we applied COMODO to study coexpression conservation between the two well-studied bacteria Escherichia coli and Bacillus subtilis. COMODO is available at: http://homes.esat.kuleuven.be/∼kmarchal/Supplementary_Information_Zarrineh_2010/comodo/index.html

Characterization of FUS Mutations in Amyotrophic Lateral Sclerosis Using RNA-Seq

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.ALS Foundation NetherlandsAdessium FoundationSeventh Framework Programme (European Commission) (grant number 259867)Thierry Latran FoundationNational Institutes of Health (U.S.) (NIH/NINDS grant R01NS073873)National Institute of Neurological Disorders and Stroke (U.S.) (NIH/NINDS grant numbers 1R01NS065847

80Se(n,?) cross-section measurement at CERN n TOF

Radiative neutron capture cross section measurements are of fundamental importance for the study of the slow neutron capture (s-) process of nucleosynthesis. This mechanism is responsible for the formation of most elements heavier than iron in the Universe. Particularly relevant are branching nuclei along the s-process path, which are sensitive to the physical conditions of the stellar environment. One such example is the branching at $^{79}$Se (3.27 × 10$^{5}$ y), which shows a thermally dependent β-decay rate. However, an astrophysically consistent interpretation requires also the knowledge of the closest neighbour isotopes involved. In particular, the $^{80}$Se(n,γ) cross section directly affects the stellar yield of the "cold" branch leading to the formation of the s-only $^{82}$Kr. Experimentally, there exists only one previous measurement on $^{80}$Se using the time of flight (TOF) technique. However, the latter suffers from some limitations that are described in this presentation. These drawbacks have been significantly improved in a recent measurement at CERN n TOF. This contribution presents a summary of the latter measurement and the status of the data analysis