Structural insights into the synthesis of FMN in prokaryotic organisms

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyl transferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.This work has been supported by MINECO, Spain (BIO2013-42978-P to MM and BFU2014-59389-P to JAH), the Aragonian Government-FEDER (B18), Autonomous Community of Madrid (S2010/BMD-2457), Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) and Universidad Industrial de Santander (project 1818 to IL).Peer Reviewe

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

Inventory of African desert dust events in the north-central Iberian Peninsula in 2003–2014 based on sun-photometer–AERONET and particulate-mass–EMEP data

A reliable identification of desert dust (DD) episodes over north-central Spain is carried out based on the AErosol RObotic NETwork (AERONET) columnar aerosol sun photometer (aerosol optical depth, AOD, and Ångström exponent, <i>α</i>) and European Monitoring and Evaluation Programme (EMEP) surface particulate-mass concentration (PM_<i>x</i>, <i>x</i> = 10, 2.5, and 2.5–10 µm) as the main core data. The impact of DD on background aerosol conditions is detectable by means of aerosol load thresholds and complementary information provided by HYSPLIT (Hybrid Single Particle Lagrangian Integrated Trajectory Model) air mass back trajectories, MODIS (Moderate Resolution Imaging Spectroradiometer) images, forecast aerosol models, and synoptic maps, which have been carefully reviewed by a human observer for each day included in the DD inventory. This identification method allows the detection of low and moderate DD intrusions and also of mixtures of mineral dust with other aerosol types by means of the analysis of <i>α</i>. During the period studied (2003–2014), a total of 152 DD episodes composed of 418 days are identified. Overall, this means ∼ 13 episodes and ∼ 35 days per year with DD intrusion, representing 9.5 % days year⁻¹. During the identified DD intrusions, 19 daily exceedances over 50 µg m⁻³ are reported at the surface. The occurrence of DD event days during the year peaks in March and June, with a marked minimum in April and lowest occurrence in winter. A large interannual variability is observed showing a statistically significant temporal decreasing trend of ∼ 3 days year⁻¹. The DD impact on the aerosol climatology is addressed by evaluating the DD contribution in magnitude and percent (in brackets) for AOD, PM₁₀, PM_2.5, and PM_{2.5 − 10}, obtaining mean values of 0.015 (11.5 %), 1.3 µg m⁻³ (11.8 %), 0.55 µg m⁻³ (8.5 %) and 0.79 µg m⁻³ (16.1 %), respectively. Annual cycles of the DD contribution for AOD and PM₁₀ present two maxima – one in summer (0.03 and 2.4 µg m⁻³ for AOD in June and PM₁₀ in August) and another in March (0.02 for AOD and 2.2 µg m⁻³ for PM₁₀) – both displaying a similar evolution with exceptions in July and September. The seasonal cycle of the DD contribution to AOD does not follow the pattern of the total AOD (close to a bell shape), whereas both PM₁₀ cycles (total and DD contribution) are more similar to each other in shape, with an exception in September. The interannual evolution of the DD contribution to AOD and PM₁₀ has evidenced a progressive decrease. This decline in the levels of mineral dust aerosols can explain up to 30 % of the total aerosol load decrease observed in the study area during the period 2003–2014. The relationship between columnar and surface DD contribution shows a correlation coefficient of 0.81 for the interannual averages. Finally, synoptic conditions during DD events are also analysed, observing that the north African thermal low causes most of the events ( ∼  53 %). The results presented in this study highlight the relevance of the area studied since it can be considered representative of the clean background in the western Mediterranean Basin where DD events have a high impact on aerosol load levels

The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements

Prokaryotic bifunctional FAD synthetases (FADSs) catalyze the biosynthesis of FMN and FAD, whereas in eukaryotes two enzymes are required for the same purpose. FMN and FAD are key cofactors to maintain the flavoproteome homeostasis in all type of organisms. Here we shed light to the properties of the hitherto unstudied bacterial FADS from the human pathogen Streptococcus pneumoniae (SpnFADS). As other members of the family, SpnFADS catalyzes the three typical activities of prokaryotic FADSs: Riboflavin kinase (RFK), ATP:FMN:adenylyltransferase (FMNAT), and FAD pyrophosphorylase (FADpp). However, several SpnFADS biophysical properties differ from those of other family members. In particular; i) the RFK activity is not inhibited by the riboflavin (RF) substrate, ii) the FMNAT and FADSpp activities require flavin substrates in the reduced state, iii) binding of adenine nucleotide ligands is required for the binding of flavinic substrates/products and iv) the monomer is the preferred state. Collectively, our results add interesting mechanistic differences among the few prokaryotic bifunctional FADSs already characterized, which might reflect the adaptation of the enzyme to relatively different environments. In a health point of view, differences among FADS family members provide us with a framework to design selective compounds targeting these enzymes for the treatment of diverse infectious diseases