Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473

The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation

The Baeyer\u2013Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer\u2013Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 \uc5 resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme\u2019s chemospecificity, to a location above the flavin\u2013peroxide adduct where catalysis occurs.Peer reviewed: YesNRC publication: Ye

Wildcat scats: Taphonomy of the predator and its micromamal prey

Small sized felids, such as wild and domestic cats, are one of the most common predators in the nature and in sites occupied by humans in archaeological and historical contexts. Wildcats have ingestion/ digestion traits highly destructive for their prey, i.e.: teeth to chew causing extreme breakage, and digestion along the entire digestive tract with low pH gastric juices causing extreme bone corrosion. Small sized cats are also well known to play with the prey and select skeletal parts to ingest. The present study is focused on the taphonomic analysis of micromammal remains recovered from scats produced by European wildcats (Felis silvestris silvestris) during several months and years. Excrements were collected from the Montes do Invernadeiro Natural Park (Galicia, Spain). Following the taphonomic methodological protocols, anatomical representation, bone breakage and degrees of digestive corrosion were evaluated. Results obtained from the skeletal representation indicate a high abundance of cranial skeleton and a high fracture pattern. Most dental elements show digestion, reaching extreme grades of corrosion, but the proportions of micromammal prey elements affected varies between taxonomic groups. The large sample size collected during 2 years, including different seasons, show no differences in either taxonomic composition or degree of digestion between the four seasons. The scat contents provide a basic and general pattern of alterations caused by carnivorous mammals on the bony elements of their prey. Some differences between felid predator species have also been observed, thus pointing out the necessity of more actualistic taphonomic analysis that will allow us to obtain a more accurate indication of modifications produced by small carnivorous mammals and specific differences.Fieldwork and sample collection has been funded by the research project USEK, nº-7-2004-05 of the SEK University of Segovia, Spain and the project. The taphonomic study was financed by the CGL2016-79334-P of the Spanish Ministry of Research

Understanding emotions: origins and roles of the amygdala

Emotions arise from activations of specialized neuronal populations in several parts of the cerebral cortex, notably the anterior cingulate, insula, ventromedial prefrontal, and subcortical structures, such as the amygdala, ventral striatum, putamen, caudate nucleus, and ventral tegmental area. Feelings are conscious, emotional experiences of these activations that contribute to neuronal networks mediating thoughts, language, and behavior, thus enhancing the ability to predict, learn, and reappraise stimuli and situations in the environment based on previous experiences. Contemporary theories of emotion converge around the key role of the amygdala as the central subcortical emotional brain structure that constantly evaluates and integrates a variety of sensory information from the surroundings and assigns them appropriate values of emotional dimensions, such as valence, intensity, and approachability. The amygdala participates in the regulation of autonomic and endocrine functions, decision-making and adaptations of instinctive and motivational behaviors to changes in the environment through implicit associative learning, changes in short- and long-term synaptic plasticity, and activation of the fight-or-flight response via efferent projections from its central nucleus to cortical and subcortical structures

Prenatal development of the human entorhinal cortex

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP-expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders