Mining the Flavoproteome of Brucella ovis, the Brucellosis causing agent in Ovis aries

Flavoproteins are a diverse class of proteins that are mostly enzymes and contain as cofactors flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which enable them to participate in a wide range of physiological reactions. We have compiled 78 potential proteins building the flavoproteome of Brucella ovis (B. ovis), the causative agent of ovine brucellosis. The curated list of flavoproteins here reported is based on (i) the analysis of sequence, structure and function of homologous proteins, and their classification according to their structural domains, clans, and expected enzymatic functions; (ii) the constructed phylogenetic trees of enzyme functional classes using 19 Brucella strains and 26 pathogenic and/or biotechnological relevant alphaproteobacteria together with B. ovis; and (iii) the evaluation of the genetic context for each entry. Candidates account for;2.7% of the B. ovis proteome, and 75% of them use FAD as cofactor. Only 55% of these flavoproteins belong to the core proteome of Brucella and contribute to B. ovis processes involved in maintenance activities, survival and response to stress, virulence, and/or infectivity. Several of the predicted flavoproteins are highly divergent in Brucella genus from revised proteins and for them it is difficult to envisage a clear function. This might indicate modified catalytic activities or even divergent processes and mechanisms still not identified. We have also detected the lack of some functional flavoenzymes in B. ovis, which might contribute to it being nonzoonotic. Finally, potentiality of B. ovis flavoproteome as the source of antimicrobial targets or biocatalyst is discussed. IMPORTANCE Some microorganisms depend heavily on flavin-dependent activities, but others maintain them at a minimum. Knowledge about flavoprotein content and functions in different microorganisms will help to identify their metabolic requirements, as well as to benefit either industry or health. Currently, most flavoproteins from the sheep pathogen Brucella ovis are only automatically annotated in databases, and only two have been experimentally studied. Indeed, certain homologues with unknown function are not characterized, and they might relate to still not identified mechanisms or processes. Our research has identified 78 members that comprise its flavoproteome, 76 of them flavoenzymes, which mainly relate to bacteria survival, virulence, and/or infectivity. The list of flavoproteins here presented allows us to better understand the peculiarities of Brucella ovis and can be applied as a tool to search for candidates as new biocatalyst or antimicrobial targets

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

Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri

We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5¿Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen

An efficient method for enzyme immobilization evidenced by atomic force microscopy

Immobilization of proteins in a functionally active form and proper orientation is fundamental for effective surface-based protein analysis. A new method is presented for the controlled and oriented immobilization of ordered monolayers of enzymes whose interaction site had been protected using the protein ligand. The utility of this method was demonstrated by analyzing the interactions between the enzyme ferredoxin-NADP+ reductase (FNR) and its redox partner ferredoxin (Fd). The quality of the procedure was deeply evaluated through enzymatic assays and atomic force microscopy. Single-molecule force spectroscopy revealed that site-specifically targeted FNR samples increased the ratio of recognition events 4-fold with regard to the standard randomly modified FNR samples. The results were corroborated using the cytochrome c reductase activity that gave an increase on surface between 6 and 12 times for the site-specifically targeted FNR samples. The activity in solution for the enzyme labeled from the complex was similar to that exhibited by wild-type FNR while FNR randomly tagged showed a 3-fold decrease. This indicates that random targeting protocols affect not only the efficiency of immobilized proteins to recognize their ligands but also their general functionality. The present methodology is expected to find wide applications in surface-based protein–protein interactions biosensors, single-molecule analysis, bioelectronics or drug screening

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

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ¿ABC, which lacks helices aA, aB and aC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered

Identification of inhibitors targeting ferredoxin-NADP+ reductase from the xanthomonas citri subsp. Citri phytopathogenic bacteria

Ferredoxin-NADP(H) reductases (FNRs) deliver NADPH or low potential one-electron donors to redox-based metabolism in plastids and bacteria. Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker disease that affects commercial citrus crops worldwide. The Xcc fpr gene encodes a bacterial type FNR (XccFPR) that contributes to the bacterial response to oxidative stress conditions, usually found during plant colonization. Therefore, XccFPR is relevant for the pathogen survival and its inhibition might represent a strategy to treat citrus canker. Because of mechanistic and structural differences from plastidic FNRs, XccFPR is also a potential antibacterial target. We have optimized an activity-based high-throughput screening (HTS) assay that identifies XccFPR inhibitors. We selected 43 hits from a chemical library and narrowed them down to the four most promising inhibitors. The antimicrobial effect of these compounds was evaluated on Xcc cultures, finding one with antimicrobial properties. Based on the functional groups of this compound and their geometric arrangement, we identified another three XccFPR inhibitors. Inhibition mechanisms and constants were determined for these four XccFPR inhibitors. Their specificity was also evaluated by studying their effect on the plastidic Anabaena PCC 7119 FNR, finding differences that can become interesting tools to discover Xcc antimicrobials

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs