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.

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.

The Biosynthesis of Flavin Cofactors in Listeria monocytogenes.

Listeria monocytogenes is riboflavin auxotrophic, but it has two genes envisaged to transform riboflavin into FMN and FAD after its uptaked by specialized transporters. One encodes a bifunctional type I FAD synthase (FADS, herein LmFADS-1), while the other produces a protein similar to type I at the FMN:ATP adenylyltransferase (FMNAT) site but with a shorter C-terminal that lacks any riboflavin kinase (RFK) motif. This second protein is rare among bacteria and has been named FADS type II (LmFADS-2). Here we present a biochemical and biophysical study of LmFADS-1 and LmFADS-2 by integrating kinetic and thermodynamic data together with sequence and structural prediction methods to evaluate their occurrence in Listeria, as well as their function and molecular properties. Despite LmFADS-1 similarities to other type I FADSs, (i) its RFK activity has not riboflavin substrate inhibition and occurs under reducing and oxidizing conditions, (ii) its FMNAT activity requires strong reducing environment, and (iii) binding of reaction products, but not substrates, favors binding of the second ligand. LmFADS-2 produces FAD under oxidizing and reducing environments, but its C-terminus module function remains unknown. Listeria species conserve both FADSs, being sequence identity high within L. monocytogenes strains. Our data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis, envisaging that in Listeria two FADSs might be required to fulfill the supply of flavin cofactors under niches that can go from saprophytism to virulence. As FADSs are attractive antimicrobial targets, understanding of FADSs traits in different species is essential to help in the discovery of specific antimicrobials.

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 α1c 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 α1c 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.

Multiskills training of medical doctors in India: Experience from Rajasthan.

BACKGROUND: The Government of India has been conducting multiskills training programmes to address the shortage of specialized human resources in gynaecology and anaesthesia for maternal and child healthcare. There has been little evaluation of the operation of these programmes, though several years have passed since their introduction. METHODS: We did a cross-sectional study to assess some aspects of the multiskills training programmes in gynaecology and anaesthesia, and the utilization of human resources trained under this scheme in Rajasthan. The analysis was primarily based on a review of records of postings of doctors obtained from the Department of Health and Family Welfare, Government of Rajasthan. Information was also obtained through qualitative interviews with 122 doctors from 14 districts of the state, irrespective of their training status. RESULTS: In 2012, a total of 302 anaesthetists and 480 gynaecologists were posted in various public health facilities in Rajasthan. Of these, 128 (42%) anaesthetists and 69 (14.4%) gynaecologists had received multiskills training. However, only 57% of trained doctors were posted at health facilities for which they were trained. The acceptance of multiskills training among doctors was found to be low. CONCLUSION: Posting and deployment of personnel who had received multiskills training was often inappropriate, leading to suboptimal utilization of the skills acquired during such training and suboptimal delivery of public health services. Accreditation of the multiskills training programmes by regulatory bodies such as the Medical Council of India may improve the acceptance of such training among MBBS doctors and their colleagues. There is a need to review multiskills training programmes.

Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity.

Prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with ATP:riboflavin kinase (RFK) activity, and the N-terminus with ATP:FMN adenylyltransferase (FMNAT) activity. The FADS from Corynebacterium ammoniagenes, CaFADS, forms transient oligomers during catalysis. These oligomers are stabilized by several interactions between the RFK and FMNAT sites from neighboring protomers, which otherwise are separated in the monomeric enzyme. Among these inter-protomer interactions, the salt bridge between E268 at the RFK site and R66 at the FMNAT-module is particularly relevant, as E268 is the catalytic base of the kinase reaction. Here we have introduced point mutations at R66 to analyze the impact of the salt-bridge on ligand binding and catalysis. Interestingly, these mutations have only mild effects on ligand binding and kinetic properties of the FMNAT-module (where R66 is located), but considerably impair the RFK activity turnover. Substitutions of R66 also modulate the ratio between monomeric and oligomeric species and modify the quaternary arrangement observed by single-molecule methods. Therefore, our data further support the cross-talk between the RFK- and FMNAT-modules of neighboring protomers in the CaFADS enzyme, and establish the participation of R66 in the modulation of the geometry of the RFK active site during catalysis.

History of religious delusions and psychosocial functioning among Mexican patients with paranoid schizophrenia.

The association between global functionality and religiosity among patients from developing and predominantly Catholic countries warrants attention. To compare religiosity and psychosocial functioning in Mexican schizophrenia patients with and without a history of religious delusions, seventy-four patients with paranoid schizophrenia were recruited. Patients with a history of religious delusions had more psychiatric hospitalizations and poorer psychosocial functioning compared with those without a history of religious delusions. No differences emerged between groups in the total scores of religiosity scales. A history of religious delusions rather than religiosity itself may have an influence on psychosocial functioning among Mexican patients with schizophrenia.

Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level.

Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg(2+) particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.

Evolutionary divergence of chloroplast FAD synthetase proteins.

BACKGROUND: Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. RESULTS: Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. CONCLUSIONS: A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity.

Evaluación de las aplicaciones locales de una solución limpiadora suave con Dinafitol de Bardana y pH8: estudio realizado en consulta privada / Evaluation of the local application of a mild cleansing solution: study carried out in private consultation

Estudio realizado en consulta privada de los 17 médicos participantes, en 150 pacientes con vulvovaginitis, de los que se reportan 148 casos provenientes de tres principales ciudades del Ecuador (Guayaquil, Quito y Cuenca).Tipo de estudio: Abierto, multicéntrico, no comparativo.Objetivo: Evaluar la eficacia y tolerancia de una solución limpiadora suave con contenido de Bardana, en el período previo al tratamiento específico, como ayuda en el control del prurito y eritema presentes en las vulvovaginitis.Material y método: Se utilizó una solución limpiadora suave con contenido de Dinafitoles de Bardana y nivel de pH 8. Fueron reclutadas 150 pacientes de acuerdo a los criterios de inclusión, no inclusión y exclusión. La sintomatología fue evaluada al inicio y al final del estudio. Se recomendó el uso del producto en higiene local dos veces al día por siete días mientras se esperaba el resultado del examen cito bacteriológico vaginal.Resultados:•98.0% de mejoría y desaparición del prurito.•96.5% de mejoría y desaparición del eritema.En cuanto a la tolerancia al producto utilizado, el 95.3% reportó la calificación de “buena”.Conclusión: Se logró demostrar la utilidad de la solución limpiadora suave con pH8 y contenido de Dinafitol de Bardana, concluyendo que su utilización está justificada en los casos de vulvovaginitis y que, gracias a la tolerancia y aceptación de las pacientes se puede recomendar también su uso cotidiano en la higiene íntima y corporal. Por los resultados obtenidos, podemos considerar el uso de esta solución limpiadora como de primera elección en el manejo de las patologías relacionadas con el prurito y eritema y como un excelente coadyuvante en el tratamiento etiológico de las vulvovaginitis en las que con frecuencia advertimos la presencia de esta sintomatología.

Study carried out in the private consultation of 17 doctors selected to participate in the study that included 150 patients with vulvovaginitis, of which 148 cases are reported from in the three main cities of the Ecuador, (Guayaquil, Quito and Cuenca). Type of study: Open, multicentral, not comparative. Objective: To evaluate the effectiveness and tolerance of a mild cleansing solution, containing Bardana, during the period prior to a specific treatment, in order to help the control of pruritus and erythema present in the vulvovaginitis. Material and method: A mild cleansing solution with a pH level 8 was used. 150 patients were recruited according to the inclusion and exclusion criteria of the study. The symptoms were evaluated at the beginning and at the end of the study. The use of the product was recommended twice a day for seven days while expecting the results of the bacteriological vaginal exam.Results:•98.0% of improvement and disappearance of pruritus.•96.5% of improvement and disappearance of erythema. As for the tolerance to the use of product, 95.3% of the patients reported it as “good”. Conclusion: It was possible to demonstrate the how important is the use of the mild cleansing solution with a pH 8. We conclude that its use is justified in the case of vulvovaginitis and that thanks to its tolerance and the patients' acceptance, it may also be recommended to be used daily in the intimate and corporal hygiene. With the reported results, one can consider the use of this mild cleansing solution as the first election in the handling of pathologies related with pruritus and erythema and as an excellent help for etiological treatment of the vulvovaginitis in those that frequently notice the presence of this symptoms.

Lactoferrin and host defense.

Lactoferrin is a multifunctional member of the transferrin family of nonheme iron-binding glycoproteins. Lactoferrin is found at the mucosal surface where it functions as a prominent component of the first line of host defense against infection and inflammation. The protein is also an abundant component of the specific granules of neutrophils and can be released into the serum upon neutrophil degranulation. While the iron-binding properties were originally believed to be solely responsible for the host defense properties ascribed to lactoferrin, it is now known that other mechanisms contribute to the broad spectrum anti-infective and anti-inflammatory roles of this protein. In this article, current information on the functions and mechanism of action of lactoferrin are reviewed, with particular emphasis on the activities that contribute to this protein's role in host defense. In addition, studies demonstrating that lactoferrin inhibits allergen-induced skin inflammation in both mice and humans, most likely secondary to TNF-alpha (tumor necrosis factor alpha) production, are summarized. Collectively, these results suggest that lactoferrin functions as a key component of mammalian host defense at the mucosal surface.