Specific features for the competent binding of substrates at the FMN adenylyltransferase site of FAD synthase from corynebacterium ammoniagenes

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN: adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN-and FAD-reacting phosphates in optimal geometry for catalysis

Insights into the fmnat active site of fad synthase: Aromaticity is essential for flavin binding and catalysis

The last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT modules of bifunctional FAD synthases (FADS) in Prokarya belong to different structural families with dissimilar chemistry and binding modes for the substrates. In this study, we analyzed the relevance of the hydrophobic environment of the flavin isoalloxazine in the FMNAT active site of Corynebacterium ammoniagenes FADS (CaFADS) through the mutational analysis of its F62, Y106, and F128 residues. They form the isoalloxazine binding cavity and are highly conserved in the prokaryotic FADS family. The spectroscopic, steady-state kinetics and thermodynamic data presented indicate that distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and, as a consequence, decreases the efficiency of the FMNAT activity. Therefore, the side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in CaFADS. The introduced mutations also modulate the activity occurring at the riboflavin kinase (RFK) module of CaFADS, further evidencing the formation of quaternary assemblies during catalysis

Diez años de selección de toros jóvenes en el centro de testaje de la raza Bruna dels Pirineus

publishedTomo I . Sección: Sistemas Ganaderos-Economía y Gestión. Sesión: Vacuno carne II. Ponencia nº

Association between olfactory dysfunction and mood disturbances with objective and subjective cognitive deficits in long-COVID

Background and purposeThe coronavirus disease 2019 (COVID-19) has been associated with olfactory dysfunction. The persistent symptoms of anosmia or hyposmia were associated in previous studies with the development of memory impairment and mood disturbances. We aimed to investigate the association between the chronicity of reported olfactory dysfunction and subjective and objective cognitive performance in long-COVID patients and to explore whether their emotional symptoms are related to their cognition.MethodsOne hundred twenty-eight long-COVID participants were recruited. Reported symptomatology, subjective memory complaints, anxiety and depression symptomatology, and trait-anxiety were assessed. Subjective memory complaints and mood disturbances were compared among groups of participants with olfactory dysfunction as an acute (AOD), persistent (POD), or nonexistent (NOD) symptom. Seventy-six of the volunteers also participated in a face-to-face session to assess their objective performance on tests of general cognitive function and verbal declarative memory. Objective cognitive performance and mood disturbances were compared among the AOD, POD, and NOD groups.ResultsThe subjective memory complaints and the anxiety and depression symptoms were similar among the groups, but the score in general cognitive function was lower in the participants with symptoms of acute olfactory dysfunction than in those with no olfactory symptoms at any time. Participants’ memory complaints were positively related to their emotional symptoms. The relationship between depressive symptomatology and memory complaints interacted with the olfactory dysfunction, as it only occurred in the participants without symptoms of olfactory dysfunction. Depressive symptomatology and acute olfactory symptoms were negatively associated with general cognitive function and delayed memory performance. The months elapsed from diagnosis to assessment also predicted delayed memory performance. Anxious symptomatology was negatively associated with the immediate ability to recall verbal information in participants who did not present olfactory dysfunction in the acute phase of the infection.ConclusionOlfactory dysfunction in the acute phase of the infection by COVID-19 is related to cognitive deficits in objective tests, and mood disturbances are associated with self-reported and objective memory. These findings may contribute to further understanding the neuropsychological and emotional aspects of long-COVID

Partial enterectomy decreases somatostatin-binding sites in residual intestine of rabbits

Three weeks after partial enterectomy in the rabbit there was an increased somatostatin concentration and a decreased number of somatostatin-binding sites (without changes in the corresponding affinity values) in the cytosol of the residual intestinal tissue, except in the terminal ileum and the colon.\ud Five weeks after surgery both the somatostatin concentration and the number of somatostatin-binding sites returned towards control values.\ud These results suggest that an increase in bowel somatostatin content could lead to down-regulation of somatostatin-binding sites in the intestinal mucosa

The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix a1c of the RFK module (positions K202, E203, F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix a1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites

Leptin modulates the response of brown adipose tissue to negative energy balance: implication of the gh/igf-i axis

The growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is involved in metabolic control. Malnutrition reduces IGF-I and modifies the thermogenic capacity of brown adipose tissue (BAT). Leptin has effects on the GH/IGF-I axis and the function of BAT, but its interaction with IGF-I and the mechanisms involved in the regulation of thermogenesis remains unknown. We studied the GH/IGF-I axis and activation of IGF-I-related signaling and metabolism related to BAT thermogenesis in chronic central leptin infused (L), pair-fed (PF), and control rats. Hypothalamic somatostatin mRNA levels were increased in PF and decreased in L, while pituitary GH mRNA was reduced in PF. Serum GH and IGF-I concentrations were decreased only in PF. In BAT, the association between suppressor of cytokine signaling 3 and the IGF-I receptor was reduced, and phosphorylation of the IGF-I receptor increased in the L group. Phosphorylation of Akt and cyclic AMP response element binding protein and glucose transporter 4 mRNA levels were increased in L and mRNA levels of uncoupling protein-1 (UCP-1) and enzymes involved in lipid anabolism reduced in PF. These results suggest that modifications in UCP-1 in BAT and changes in the GH/IGF-I axis induced by negative energy balance are dependent upon leptin levels.This research was funded by the Spanish Ministry of Science and Innovation with the help of European FEDER funding (FIS PI19/00166), Ministerio de Economía y Competitividad (BFU 2017-82565-C2-1-R) and the Network Center for Biomedical Research on Obesity and Nutrition (CIBEROBN) Instituto Carlos III. S.C. was supported by CIBEROB