Fungal peroxygenase chimera identified by sub-domain shuffling and split-gfp reveal retained activity and altered specificity

Fungal peroxygenases[1] catalyse the hydroxylation of aromatic and aliphatic C-H bonds with high activities, selectivities and thereby relying solely on hydrogen peroxide as a cofactor.[2] Although there are more than 4000 putative fungal peroxygenase genes annotated, only Agrocybe aegerita unspecific peroxygenase (AaeUPO) could be heterologously expressed in S. cerevisiae after five rounds of directed evolution and screening more than 9000 transformants.[3] Four of the identified nine mutations were located in the signal-peptide. However, as “you get what you screen for” in directed evolution, the enzyme has been evolved towards the substrate and not just on the increased expression level. Many fungal genomes carry several dozens of UPO genes suggesting they harbour different specificities and perhaps even catalytic activities. This requires a substrate unspecific screening system for the evolution of these UPOs for heterologous expression. Please click Additional Files below to see the full abstract

Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands

QCD and strongly coupled gauge theories : challenges and perspectives

We highlight the progress, current status, and open challenges of QCD-driven physics, in theory and in experiment. We discuss how the strong interaction is intimately connected to a broad sweep of physical problems, in settings ranging from astrophysics and cosmology to strongly coupled, complex systems in particle and condensed-matter physics, as well as to searches for physics beyond the Standard Model. We also discuss how success in describing the strong interaction impacts other fields, and, in turn, how such subjects can impact studies of the strong interaction. In the course of the work we offer a perspective on the many research streams which flow into and out of QCD, as well as a vision for future developments.Peer reviewe

Effort-related functions of nucleus accumbens dopamine and associated forebrain circuits

Background Over the last several years, it has become apparent that there are critical problems with the hypothesis that brain dopamine (DA) systems, particularly in the nucleus accumbens, directly mediate the rewarding or primary motivational characteristics of natural stimuli such as food. Hypotheses related to DA function are undergoing a substantial restructuring, such that the classic emphasis on hedonia and primary reward is giving way to diverse lines of research that focus on aspects of instrumental learning, reward prediction, incentive motivation, and behavioral activation. Objective The present review discusses dopaminergic involvement in behavioral activation and, in particular, emphasizes the effort-related functions of nucleus accumbens DA and associated forebrain circuitry. Results The effects of accumbens DA depletions on food-seeking behavior are critically dependent upon the work requirements of the task. Lever pressing schedules that have minimal work requirements are largely unaffected by accumbens DA depletions, whereas reinforcement schedules that have high work (e.g., ratio) requirements are substantially impaired by accumbens DA depletions. Moreover, interference with accumbens DA transmission exerts a powerful influence over effort-related decision making. Rats with accumbens DA depletions reallocate their instrumental behavior away from food-reinforced tasks that have high response requirements, and instead, these rats select a less-effortful type of food-seeking behavior. Conclusions Along with prefrontal cortex and the amygdala, nucleus accumbens is a component of the brain circuitry regulating effort-related functions. Studies of the brain systems regulating effort-based processes may have implications for understanding drug abuse, as well as energy-related disorders such as psychomotor slowing, fatigue, or anergia in depression

Preparation of aminoethyl glycosides for glycoconjugation

The synthesis of a number of aminoethyl glycosides of cell-surface carbohydrates, which are important intermediates for glycoarray synthesis, is described. A set of protocols was developed which provide these intermediates, in a short number of steps, from commercially available starting materials

Iron-Catalyzed C—H Insertions: Organometallic and Enzymatic Carbene Transfer Reactions

C—H insertion reactions with organometallic and enzymatic catalysts based on earth-abundant iron complexes remain one of the major challenges in organic synthesis. In this report, we describe the development and application of these iron-based catalysts in the reaction of two different carbene precursors with N-heterocycles for the first time. While FeTPPCl showed excellent reactivity in the Fe(III) state with diazoacetonitrile, the highest activities of the YfeX enzyme could be achieved upon heme-iron reduction to Fe(II) with both diazoacetonitrile and ethyl diazoacetate. This highlights unexpected and subtle differences in reactivity of both iron catalysts. Deuterium labeling studies indicated a C—H insertion pathway and a marked kinetic isotope effect. This transformation features mild reaction conditions, excellent yields or turnover numbers with broad functional group tolerance, including gram-scale applications giving a unique access to functionalized N-heterocycles.</p