Identification, analysis, and modeling of the YUCCA protein family genome-wide in Coffea canephora.

Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins. YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.

The role of remote flavin adenine dinucleotide pieces in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.

Combined molecular modelling and 3D-QSAR study for understanding the inhibition of NQO1 by heterocyclic quinone derivatives.

A combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular modelling methods were used to understand the potent inhibitory NAD(P)H:quinone oxidoreductase 1 (NQO1) activity of a set of 52 heterocyclic quinones. Molecular docking results indicated that some favourable interactions of key amino acid residues at the binding site of NQO1 with these quinones would be responsible for an improvement of the NQO1 activity of these compounds. The main interactions involved are hydrogen bond of the amino group of residue Tyr128, π-stacking interactions with Phe106 and Phe178, and electrostatic interactions with flavin adenine dinucleotide (FADH) cofactor. Three models were prepared by 3D-QSAR analysis. The models derived from Model I and Model III, shown leave-one-out cross-validation correlation coefficients (q2LOO ) of .75 and .73 as well as conventional correlation coefficients (R2 ) of .93 and .95, respectively. In addition, the external predictive abilities of these models were evaluated using a test set, producing the predicted correlation coefficients (r2pred ) of .76 and .74, respectively. The good concordance between the docking results and 3D-QSAR contour maps provides helpful information about a rational modification of new molecules based in quinone scaffold, in order to design more potent NQO1 inhibitors, which would exhibit highly potent antitumor activity.

The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.

Enhanced thermal stability and pH behavior of glucose oxidase on electrostatic interaction with polyethylenimine.

Electrostatic interactions, mediated by ionic-exchange, between polyethylenimine (PEI) and glucose oxidase (GOx) were used to form GOx-PEI macro-complex, which were evaluated for pH and thermal stability of GOx. Under the experimental conditions, the complex had a dominant GOx presence on its surface and a hydrodynamic diameter of 205 ± 16 nm. Activity was evaluated from 40 to 75 °C, and at pH from 2 to 12. GOx activity in complex was maintained up to 70 °C and it was lost at 75 °C. In contrast, free GOx showed a maximum activity at 50 °C, which was completely lost at 70 °C. This difference, observed by fluorescence analysis, was associated with the compact unfolded structure of GOx in the complex. This GOx stability was not observed under pH variations, and complex formation was only possible at pH ≥ 5 where enzymatic activity was diminished by the presence of PEI.

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2.

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

The C-terminal extension of bacterial flavodoxin-reductases: involvement in the hydride transfer mechanism from the coenzyme.

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP(+) than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.

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.

Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma cruzi.

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

Entamoeba histolytica thioredoxin reductase: molecular and functional characterization of its atypical properties.

BACKGROUND: Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance. METHODS: The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR). RESULTS: Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S-nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR. CONCLUSIONS AND GENERAL SIGNIFICANCE: Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells.

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bioelectrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 µW cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 µW cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxidase bioanodes and/or biofuel cells.

Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 ß-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the ß-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the ß-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 ß-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

Molecular basis of two novel mutations found in type I methemoglobinemia.

Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek. The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild-type CYB5R3, as well as two of these novel mutations, S54R and F157C, was amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability.

Insights into the specificity of thioredoxin reductase-thioredoxin interactions. A structural and functional investigation of the yeast thioredoxin system.

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M ). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

Rat ventral prostate xanthine oxidase-mediated metabolism of acetaldehyde to acetyl radical.

Alcohol drinking is known to lead to deleterious effects on prostate epithelial cells from humans and experimental animals. The understanding of the mechanisms underlying these effects is relevant to intraprostatic ethanol treatment of benign prostatic hyperplasia and to shed some light into the conflictive results linking alcohol consumption to prostate cancer. In previous studies, we provided evidence about the presence in the rat ventral prostate of cytosolic and microsomal metabolic pathways of ethanol to acetaldehyde and 1-hydroxyethyl radical and about the low levels of alcohol dehydrogenase and aldehyde dehydrogenase. Acetaldehyde accumulation in prostate tissue and oxidative stress promotion were also observed. In this study, we report that in the ventral prostate cytosolic fraction, xanthine oxidoreductase is able to metabolize acetaldehyde to acetyl radical. The identification of the acetyl was performed by GC-MS of the silylated acetyl-PBN adduct. Reference adduct was generated chemically. Formation of acetyl was also observed using pure xanthine oxidase. The generation of acetyl by the prostate cytosol was inhibited by allopurinol, oxypurinol, diphenyleneiodonium chloride, folate, and ellagic acid. Results suggest that metabolism of ethanol to acetaldehyde and to 1-hydroxyethyl and acetyl radicals could be involved in the deleterious effects of alcohol drinking on prostate epithelial cells.

Trehalose-mediated thermal stabilization of glucose oxidase from Aspergillus niger.

Thermal inactivation and enzyme kinetics of glucose oxidase (a FAD dependent enzyme) were studied in the absence and presence of trehalose. The inactivation rate constant decreased by up to 50% at temperatures between 50 and 70 degrees C in the presence of 0.6M trehalose; as a consequence the glucose oxidase half-life increased. Intrinsic fluorescence spectra showed a maximum center of spectral mass (CSM) red shift of 6.5nm. Therefore, major structural changes seem to be related to glucose oxidase thermal inactivation. Trehalose decreased the rate constant for unfolding as monitored by CSM red shift kinetics indicating that this disaccharide favors the most compact folded state. The E(a) for unfolding was increased from 204 to 221kJ mol(-1). It is proposed that FAD dissociation is preceded by the exposition of hydrophobic regions, while the presence of trehalose was able to hinder the release of FAD. Enzyme kinetics analysis showed that trehalose does not affect V(max) but instead decreases K(m); as a result enzyme efficiency was increased. The stabilizing effect of trehalose in a cofactor-dependent enzyme has not been tested to date. In addition, glucose oxidase has an enormous commercial importance and therefore, the use of trehalose to stabilize glucose oxidase in its multiple applications seems to be promising.

Wired-enzyme core-shell Au nanoparticle biosensor.

We report a fully integrated core-shell nanoparticle system responsive to glucose. The system is comprised of self-assembled glucose oxidase and an osmium molecular wire on core-shell Au nanoparticles. Characterization of the functional nanoparticles by spectroscopy, quartz crystal microbalance and electrochemical techniques has shown that the catalytically active shell has a structure as designed and all components are active in the self-assembled multilayer shell. Furthermore, amperometric reagentless detection of glucose and contactless photonic biosensing by the Os(II) resonant Raman signal have been demonstrated. The enzymatic reduction of FAD by glucose and further reduction of the Raman silent Os(III) by FADH 2 yields a characteristic enzyme-substrate calibration curve in the millimolar range. Furthermore, coupling of electronic resonant Raman of the osmium complex with the SERS amplification by Au NPs plasmon resonance has been demonstrated which leads to an extra enhancement of the biosensor signal. We present a proof of concept extending the work done with planar surfaces to core-shell NPs as an advance in the design of glucose-responsive chemistry detected by SERS-like methods.

Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+.

BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans.

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 angstroms resolution at a synchrotron source.