BACKGROUND
ferredoxin-nadp reductases  are flavoenzymes that catalyze electron transfer between nadp and the iron-sulfur protein ferredoxin  or flavin mononucleotide-containing flavodoxin  <cit> .
these enzymes are normally present as monomeric proteins in plastids, bacteria, mitochondria, and apicoplasts of intracellular parasites, where they catalyze the reaction described in equation , using noncovalently bound fad as a prosthetic group.
<dig> fd + nadp+ + h+⇆ <dig> fd + nadph
depending on the primary energy source of the organism the reaction catalyzed by fnr can be driven toward one of the directions indicated by the double arrow in equation .
in heterotrophic bacteria and eukaryotes, the catalysis is driven toward ferredoxin reduction providing reducing power for a wide range of metabolic pathways  <cit> .
structural information is available for fnrs from plastids , cyanobacteria  and bacteria .
topologically, there is a number of similarities within the two structural domains of fnrs of a different origin.
the protein core, composed of a αβ-sandwich with a 5-strand β-sheet surrounded by  <dig> α-helices, is strongly conserved among fnrs.
structural differences however, appear in the size and conformation of the loops on the protein surface  <cit> .
fad binding site is located between the protein domains and has been described in detail by bruns & karplus  <cit> .
the binding of the prosthetic group to fnrs from different sources is structurally different.
the crystal structures of enzymes from plastids and cyanobacteria have fad molecule in an extended conformation, while those from others prokaryotes are bended toward themselves and establish hydrogen bond interactions between the adenine and the isoalloxazine  <cit> .
sequence alignments of fnrs from different organisms revealed conservation profiles in close agreement with above discussed structural differences.
a sequence alignment of plants, bacteria and algae fnrs reveals that the overall homology between enzyme primary structures is not so high, although amino acid sequence conservation between plant enzymes, for example, is significantly better .
leptospira interrogans is a spirochaete parasitic bacterium known to infect humans and mammals including cattle, dogs, pigs, horses and its natural carrier hosts, rodents.
quite surprisingly, according to phylogenetic analysis, leptospira fnr  has been recently suggested to belong to the plastidic class fnrs  <cit> .
the low identity in primary structure with the members of plastidic fnrs , makes lepfnr an interesting target for structural and biochemical analysis aiming to better understand the structural grounds of the high catalytic efficiency of these oxidoreductases.
in addition, the structure of lepfnr provides structural basis for the design of efficient and specific drugs dedicated to leptospirosis treatment.
in this work we submitted lepfnr to both structural and biochemical analysis.
the crystal structures of lepfnr, presented here, reveal the molecular basis of recognition and binding of both prosthetic group fad and substrate nadp+ by the enzyme.
structural comparison of lepfnr with other fnrs, both from plant and bacterial origin, demonstrated more structural similarities of lepfnr with plant enzymes than with the bacterial fnrs.
small angle scattering experiments prove that lepfnr is a monomeric enzyme in solution.
finally, our biochemical data indicate that the lepfnr has a high enzymatic activity similar to those reported for the plastidic class enzymes and, considerably different from bacterial flavoenzymes which display lower turnover rates.
RESULTS
overall quality of the x-ray models
the quality of the lepfnr crystal structure, refined to  <dig>  Å resolution, is fine as judged by stereochemical and refinement parameters .
overall topology of the lepfnr molecule has the canonical fnr fold  <cit> , which consists of two well defined domains, although several unique structural features were found in lepfnr 3d structure.
the n-terminal domain forms a 6-stranded antiparallel β-barrel, and harbors the fad binding site.
the c-terminal domain folds as a αβ-sandwich with a 5-stranded β-sheet surrounded by six α-helices .
a unique feature present in lepfnr structure is the small helix formed in the loop 76– <dig> .
this region has been previously identified as an important docking site of ferredoxin binding, mediated by its positively charged amino acid residues  <cit> .
although being exposed to solvent, this protein-protein recognition site has low mobility as judged from b-factors analyses .
highly flexible regions of the protein are mostly confined to the surface loops .
values in brackets represent the values for each parameter in the highest resolution shell.
rmerge = Σhkl Σi ii - ⟨i⟩/Σhkl Σi ii
rpim = Σhkl 1/ <dig> Σi|ii - ⟨i⟩|/Σhkl Σi ii, where n is the redundancy measured.
two out of the four molecules present in asymmetric unit of lepfnr crystal jointly coordinate a metal ion via two glutamates and one histidine residues, as shown in figure 2b.
this metal binding site is unique and has not been previously observed in any other fnr crystal structures.
fnrs are known to function as monomeric proteins  <cit> , however it has been recently suggested that two fnr isoforms from arabidopsis thaliana are capable of dimer formation  <cit> .
thus, we decided to investigate the oligomeric state of lepfnr in solution by small angle x-ray scattering.
lepfnr solution scattering
x-ray scattering curves at the protein concentration of  <dig> mg/ml and  <dig> mg/ml have essentially the same profile; therefore we used scattering data measured at  <dig> mg/ml to benefit from their better statistics and resolution.
comparison of saxs data with crystallographic structures of lepfnr monomers and dimers using crysol reveals that lepfnr forms monomers in solution .
analysis of the distance-distribution p function  led us to conclude that the protein has a maximum dimension dmax of  <dig>  ±  <dig>  nm, compatible with a fnr monomer.
the value of  <dig>  ±  <dig>  nm for rg calculated from the distance distribution function is in a good agreement with the estimate derived from the guinier analysis of  <dig>  nm.
furthermore, a structural superposition of the ab initio low-resolution envelope of the protein with the high-resolution x-ray structure of the lepfnr monomer  leaves no doubt that the protein is indeed a monomer in solution.
* exp., calculated from the experimental data at  <dig> mg/ml.
† mod, parameters obtained from the lepfnr monomer crystallographic structure
‡ dam, parameters of the averaged dummy atom model over  <dig> models.
€ shannon channels number ns = /π
π model residues number
χ parameter of comparison with experimental data
¶resolution = 2*π/qmáx
η mw obtained by comparison with standard protein bsa
our results on lepfnr monomeric form obtained from solution x-ray scattering diverge from the results of the recent crystallographic studies of plasmodium falciparum fnr  <cit> .
in the latter protein, two oxidized cysteine residues form a covalent bond joining two molecules in a dimer.
the cysteine residues involved in the bridge is highly conserved over the plasmodial fnrs, but not in plant and homologous fnrs  <cit> .
lepfnr, on the other hand, exhibits higher sequential identity with plant fnrs and does not contain cysteine residues in the position required to form the intermolecular disulfide bridge.
this analysis strongly suggests that metal mediated lepfnr dimerization, observed in the crystal structure, is induced by crystallization effects and do not represent a physiologically relevant oligomeric state of the protein.
fad binding site
crystal structure analysis reveals that unlike in usual prokaryote fnrs, lepfnr accommodates the fad molecule in an extended conformation between the two enzyme domains .
adenosine portion of fad is less restrained and more disordered as could be concluded from lepfnr crystallographic b-factors analysis.
an increased disorder of adenosine portion of fad as compared to the whole molecule is common for plastidic class fnrs  <cit> .
the flavin group of fad is tightly bound to fnr binding cleft through strong hydrogen bonds .
the main chain atoms of ser <dig>  ile <dig>  lys <dig> and leu <dig> interact directly with flavin moiety creating the core of the binding site .
ser <dig> and tyr <dig> also interact to flavin group via their side chains, adding to a stereochemical environment able to bind and tightly fix the fad molecule.
it is interesting to note that although ile <dig> plays an important role in flavin recognition and interacts with the flavin group through a strong hydrogen bond , this residue is not conserved in fnrs .
similarly, lys <dig> is only partially conserved in fnr family and is present in approximately half of the fnrs shown in the alignment .
crystal structure analysis reveals, however, that both residues participate in fad recognition and binding through their main chain groups, and, therefore, evolutionary restraints on the specific amino acid type have been lost at these positions.
the adenosine group also participates in a rich hydrogen bond network through its pyrophosphate portion.
cys <dig>  arg <dig>  ser <dig> and val <dig> are at hydrogen bonding distances to pyrophosphate group strongly anchoring it to lepfnr cleft.
this results in a low mobility of adenosine group in lepfnr structure.
adenosine ribose is also involved in strong hydrogen bond interaction with asp <dig> as judged from a short  <dig>  Å length of this bond.
this is a particular feature of lepfnr since this residue is not conserved over fnr family.
at variance, this aspartate is replaced by a hydrophobic leucine residue in plastidic fnrs and by an alanine in some bacterial fnrs, so that this additional hydrogen bond in adenosine binding pocket appears to be new in fnr structures.
the phenol ring of the carboxyl terminal tyrosine  and the flavin group are practically coplanar maximizing π-orbital overlapping, as have been observed in all plastidic class fnrs  <cit> , resulting in a large portion of the isoalloxazine moiety shielded from the bulk solution.
this structural feature, together with the extended fad conformation, has been implicated in the high catalytic efficiencies displayed by the plastidic fnrs  <cit> .
structural comparison between plant and bacterial fnrs reveals an important and distinguished feature among them.
plastidic fnrs have a common motif slcv.
in lepfnr this region is less conserved  but still contains both basic amino acids  close to the fad binding site.
in all plastidic fnrs previously known, fad is bound in an extended conformation and its adenine interacts with a tyrosine residue belonging to a typical cluster always present  <cit> .
surprisingly, in lepfnr fad acquired a different conformation in which its adenine moiety interacts with phe <dig> .
the latter amino acid is not conserved in other plastidic fnrs and resides on the protruding sheet-loop-sheet interacting with the 2'-p-amp region of fad.
this structural motif is missing in all bacterial class fnrs.
moreover, in the bacterial enzymes the basic residues are replaced by the hydrophobic isoleucine and leucine residues that accommodate the bended fad in the cleft between the n and c-terminal domains.
taken together this evidence provides an explanation why fad molecule binds to lepfnr in an extended conformation, typical for plant fnrs, and not in a bended form observed in bacterial fnrs, further highlighting structural and sequence similarities between leptospira fnr and its plant homologs.
lepfnr·nadp+ complex crystal structure
lepfnr·nadp+ crystal structure, refined to  <dig>  Å resolution, revealed moderate structural changes when compared to lepfnr crystal structure .
statistics of the model is given in table  <dig>  the high quality of electron density maps, allowed the unambiguously positioning of the nadp+ molecule noncovalently bound to the c-terminal domain of lepfnr.
small, but distinct, structural rearrangements could be identified upon nadp+ binding.
tyr <dig> changes its side-chain conformation and bends toward the nadp+ in lepfnr·nadp+ crystal structure, thus facilitating interactions between adenine phosphate and its side chain hydroxyl group.
this feature was also observed in the crystal structure of anabena fnr  <cit> .
nadp+ binding also causes changes in arg <dig> conformation, which also is part of the nadp+ binding site .
nadp+ molecule is attached to lepfnr c-terminal domain by a network of hydrogen bonds .
similarly to fad binding, the adenine portion of nadp+ is tightly bound to lepfnr.
in opposite, the nicotinamide group has an increased mobility, as indicated by crystallographic b-factors.
at atomic level, adenine moiety makes water-mediated hydrogen bonds with gly <dig>  gly <dig>  met <dig> and ile <dig> main chain atoms.
the ribose group also interacts with ser <dig> and forms water-mediated hydrogen bonds with ala <dig>  the phosphate group is anchored by the hydrogen bonds with arg <dig> and arg <dig>  all residues mentioned above have their counterparts in the already identified crystal structures of pea and anabaena fnrs complexed with nadp+  <cit> .
at variance, in lepfnr the nicotinamide portion of nadp+ makes water mediated hydrogen bonds with asp <dig>  arg <dig> and glu <dig>  lysine  <dig> also interacts to nadp+ by a hydrogen bond with oxygen of phosphate group, as indicated in figure 4b.
the overall structural adjustments induced by nadp+ binding in lepfnr structure are modest, and rmsd deviations between nadp+ bound and nadp+-free states are of  <dig>  Å as calculated for cα positions only.
structural superposition of the 3d models using their n-terminal domains shows a maximum displacement of  <dig>  Å between respective cα positions in these two models, which is confined to the c-terminal helices region.
the superposition of the structures is given in figure  <dig>
enzymatic activity
as shown in table  <dig>  lepfnr has a high diaphorase activity, reaction commonly used to characterize fnrs  <cit> .
similarly, lepfnr exhibited km for nadp+ which is identical to the one obtained for the pea  enzyme .
the kcat/km for lepfnr is circa 75% with respect to that of the latter fnr.
these steady state kinetic studies reveal that the lepfnr enzyme has high affinity for nadph.
the affinity for nadp+ was determined by difference absorption spectroscopy after successive addition of the nucleotide.
the lepfnr enzyme has a kd for nadp+ of  <dig> μm, which is in the same range of the affinities observed for the p. sativum enzyme and other reductases .
these data are in agreement with the suggested affinities for nadph deduced from steady state kinetic studies.
the different parameters were obtained as described in experimental procedures.
each parameter value represents the average of  <dig> independent experiments.
CONCLUSIONS
crystal structures of lepfnr reveal a typical plastidic type reductase, analogous in its folding architecture to the enzymes found in plant and cyanobacteria, in spite of displaying an important divergence at primary structure level.
the typical fad and nadp+ binding domains are well conserved among them and the amino acids involved in prosthetic group and nadp+ binding have a high degree of structural conservation.
our analysis reveal several distinct key features of this structure: 1) the fad molecule is bound in an extended conformation and the adenosine moiety is interacting with a different aromatic residue than in all other plastidic reductases; 2) the poorly conserved amino acid fragment 113-efiikrdn- <dig> still contains both basic amino acids that participate in the fad binding; 3) the carboxyl terminal amino acid is a tyrosine, that is planar to the isoalloxazine at  <dig>  Å distance.
all these features of the lepfnr are found in enzymatically highly efficient reductases and are in a good agreement with the biochemical data we have obtained for this enzyme.
structural comparison between present lepfnr crystal structures and the x-ray structure of the previously determined anabaena fnr complexed with nadp+  <cit>  reveals a number of important similarities in the model of substrate binding between these two enzymes.
the adenosine part of nadp+ and the 2'phosphate group of the substrate are in equivalent positions in these two structures.
in both models the nicotinamide reactive part of substrate is not positioned in a productive form and was found too far from the isoalloxazine to allow electron transfer .
the position observed for the anabaena fnr·nadp+ complex is somewhat different than the observed in lepfnr·nadp+ crystal structure.
a sequence of three steps for the coenzyme recognition and binding mechanism has been proposed to explain the different conformation obtained in the anabaena fnr·nadp+ complex  <cit> .
here, the position adopted by the nadp+ nicotinamide bound to the lepfnr enzyme may represent another intermediate before the productive binding of the substrate.
an interesting outcome of our analysis is that the binding of the substrate to holoenzyme produces not only a rearrangement of some of the residues involved in its binding but also a moderate but indubitable conformational change in the carboxyl terminal domain.
this structural rearrangement can help to explain the strong negative cooperativity that has been observed for the ferredoxin and nadp+ binding  <cit> .
atomic coordinates and structure factors were deposited to the protein data bank and received accession codes 2rc <dig> and 2rc <dig>

 ferredoxin-nadp reductases are flavoenzymes that catalyze electron transfer between nadp and the iron-sulfur protein ferredoxin or flavin mononucleotide-containing flavodoxin. these enzymes are normally present as monomeric proteins in plastids, bacteria, mitochondria, and apicoplasts of intracellular parasites, where they catalyze the reaction described in equation, using noncovalently bound fad as a prosthetic group. fd + nadp+ + h+⇆ fd + nadph depending on the primary energy source of the organism the reaction catalyzed by fnr can be driven toward one of the directions indicated by the double arrow in equation. in heterotrophic bacteria and eukaryotes, the catalysis is driven toward ferredoxin reduction providing reducing power for a wide range of metabolic pathways. structural information is available for fnrs from plastids, cyanobacteria and bacteria. topologically, there is a number of similarities within the two structural domains of fnrs of a different origin. the protein core, composed of a αβ-sandwich with a 5-strand β-sheet surrounded by α-helices, is strongly conserved among fnrs. structural differences however, appear in the size and conformation of the loops on the protein surface. fad binding site is located between the protein domains and has been described in detail by bruns & karplus. the binding of the prosthetic group to fnrs from different sources is structurally different. the crystal structures of enzymes from plastids and cyanobacteria have fad molecule in an extended conformation, while those from others prokaryotes are bended toward themselves and establish hydrogen bond interactions between the adenine and the isoalloxazine. sequence alignments of fnrs from different organisms revealed conservation profiles in close agreement with above discussed structural differences. a sequence alignment of plants, bacteria and algae fnrs reveals that the overall homology between enzyme primary structures is not so high, although amino acid sequence conservation between plant enzymes, for example, is significantly better. leptospira interrogans is a spirochaete parasitic bacterium known to infect humans and mammals including cattle, dogs, pigs, horses and its natural carrier hosts, rodents. quite surprisingly, according to phylogenetic analysis, leptospira fnr has been recently suggested to belong to the plastidic class fnrs. the low identity in primary structure with the members of plastidic fnrs, makes lepfnr an interesting target for structural and biochemical analysis aiming to better understand the structural grounds of the high catalytic efficiency of these oxidoreductases. in addition, the structure of lepfnr provides structural basis for the design of efficient and specific drugs dedicated to leptospirosis treatment. in this work we submitted lepfnr to both structural and biochemical analysis. the crystal structures of lepfnr, presented here, reveal the molecular basis of recognition and binding of both prosthetic group fad and substrate nadp+ by the enzyme. structural comparison of lepfnr with other fnrs, both from plant and bacterial origin, demonstrated more structural similarities of lepfnr with plant enzymes than with the bacterial fnrs. small angle scattering experiments prove that lepfnr is a monomeric enzyme in solution. finally, our biochemical data indicate that the lepfnr has a high enzymatic activity similar to those reported for the plastidic class enzymes and, considerably different from bacterial flavoenzymes which display lower turnover rates.  overall quality of the x-ray models the quality of the lepfnr crystal structure, refined to Å resolution, is fine as judged by stereochemical and refinement parameters. overall topology of the lepfnr molecule has the canonical fnr fold, which consists of two well defined domains, although several unique structural features were found in lepfnr 3d structure. the n-terminal domain forms a 6-stranded antiparallel β-barrel, and harbors the fad binding site. the c-terminal domain folds as a αβ-sandwich with a 5-stranded β-sheet surrounded by six α-helices. a unique feature present in lepfnr structure is the small helix formed in the loop 76–. this region has been previously identified as an important docking site of ferredoxin binding, mediated by its positively charged amino acid residues. although being exposed to solvent, this protein-protein recognition site has low mobility as judged from b-factors analyses. highly flexible regions of the protein are mostly confined to the surface loops. values in brackets represent the values for each parameter in the highest resolution shell. rmerge = Σhkl Σi ii - ⟨i⟩/Σhkl Σi ii rpim = Σhkl 1/ Σi|ii - ⟨i⟩|/Σhkl Σi ii, where n is the redundancy measured. two out of the four molecules present in asymmetric unit of lepfnr crystal jointly coordinate a metal ion via two glutamates and one histidine residues, as shown in figure 2b. this metal binding site is unique and has not been previously observed in any other fnr crystal structures. fnrs are known to function as monomeric proteins, however it has been recently suggested that two fnr isoforms from arabidopsis thaliana are capable of dimer formation. thus, we decided to investigate the oligomeric state of lepfnr in solution by small angle x-ray scattering. lepfnr solution scattering x-ray scattering curves at the protein concentration of mg/ml and mg/ml have essentially the same profile; therefore we used scattering data measured at mg/ml to benefit from their better statistics and resolution. comparison of saxs data with crystallographic structures of lepfnr monomers and dimers using crysol reveals that lepfnr forms monomers in solution. analysis of the distance-distribution p function led us to conclude that the protein has a maximum dimension dmax of ± nm, compatible with a fnr monomer. the value of ± nm for rg calculated from the distance distribution function is in a good agreement with the estimate derived from the guinier analysis of nm. furthermore, a structural superposition of the ab initio low-resolution envelope of the protein with the high-resolution x-ray structure of the lepfnr monomer leaves no doubt that the protein is indeed a monomer in solution. * exp., calculated from the experimental data at mg/ml. † mod, parameters obtained from the lepfnr monomer crystallographic structure ‡ dam, parameters of the averaged dummy atom model over models. € shannon channels number ns = /π π model residues number χ parameter of comparison with experimental data ¶resolution = 2*π/qmáx η mw obtained by comparison with standard protein bsa our results on lepfnr monomeric form obtained from solution x-ray scattering diverge from the results of the recent crystallographic studies of plasmodium falciparum fnr. in the latter protein, two oxidized cysteine residues form a covalent bond joining two molecules in a dimer. the cysteine residues involved in the bridge is highly conserved over the plasmodial fnrs, but not in plant and homologous fnrs. lepfnr, on the other hand, exhibits higher sequential identity with plant fnrs and does not contain cysteine residues in the position required to form the intermolecular disulfide bridge. this analysis strongly suggests that metal mediated lepfnr dimerization, observed in the crystal structure, is induced by crystallization effects and do not represent a physiologically relevant oligomeric state of the protein. fad binding site crystal structure analysis reveals that unlike in usual prokaryote fnrs, lepfnr accommodates the fad molecule in an extended conformation between the two enzyme domains. adenosine portion of fad is less restrained and more disordered as could be concluded from lepfnr crystallographic b-factors analysis. an increased disorder of adenosine portion of fad as compared to the whole molecule is common for plastidic class fnrs. the flavin group of fad is tightly bound to fnr binding cleft through strong hydrogen bonds. the main chain atoms of ser ile lys and leu interact directly with flavin moiety creating the core of the binding site. ser and tyr also interact to flavin group via their side chains, adding to a stereochemical environment able to bind and tightly fix the fad molecule. it is interesting to note that although ile plays an important role in flavin recognition and interacts with the flavin group through a strong hydrogen bond, this residue is not conserved in fnrs. similarly, lys is only partially conserved in fnr family and is present in approximately half of the fnrs shown in the alignment. crystal structure analysis reveals, however, that both residues participate in fad recognition and binding through their main chain groups, and, therefore, evolutionary restraints on the specific amino acid type have been lost at these positions. the adenosine group also participates in a rich hydrogen bond network through its pyrophosphate portion. cys arg ser and val are at hydrogen bonding distances to pyrophosphate group strongly anchoring it to lepfnr cleft. this results in a low mobility of adenosine group in lepfnr structure. adenosine ribose is also involved in strong hydrogen bond interaction with asp as judged from a short Å length of this bond. this is a particular feature of lepfnr since this residue is not conserved over fnr family. at variance, this aspartate is replaced by a hydrophobic leucine residue in plastidic fnrs and by an alanine in some bacterial fnrs, so that this additional hydrogen bond in adenosine binding pocket appears to be new in fnr structures. the phenol ring of the carboxyl terminal tyrosine and the flavin group are practically coplanar maximizing π-orbital overlapping, as have been observed in all plastidic class fnrs, resulting in a large portion of the isoalloxazine moiety shielded from the bulk solution. this structural feature, together with the extended fad conformation, has been implicated in the high catalytic efficiencies displayed by the plastidic fnrs. structural comparison between plant and bacterial fnrs reveals an important and distinguished feature among them. plastidic fnrs have a common motif slcv. in lepfnr this region is less conserved but still contains both basic amino acids close to the fad binding site. in all plastidic fnrs previously known, fad is bound in an extended conformation and its adenine interacts with a tyrosine residue belonging to a typical cluster always present. surprisingly, in lepfnr fad acquired a different conformation in which its adenine moiety interacts with phe. the latter amino acid is not conserved in other plastidic fnrs and resides on the protruding sheet-loop-sheet interacting with the 2'-p-amp region of fad. this structural motif is missing in all bacterial class fnrs. moreover, in the bacterial enzymes the basic residues are replaced by the hydrophobic isoleucine and leucine residues that accommodate the bended fad in the cleft between the n and c-terminal domains. taken together this evidence provides an explanation why fad molecule binds to lepfnr in an extended conformation, typical for plant fnrs, and not in a bended form observed in bacterial fnrs, further highlighting structural and sequence similarities between leptospira fnr and its plant homologs. lepfnr·nadp+ complex crystal structure lepfnr·nadp+ crystal structure, refined to Å resolution, revealed moderate structural changes when compared to lepfnr crystal structure. statistics of the model is given in table the high quality of electron density maps, allowed the unambiguously positioning of the nadp+ molecule noncovalently bound to the c-terminal domain of lepfnr. small, but distinct, structural rearrangements could be identified upon nadp+ binding. tyr changes its side-chain conformation and bends toward the nadp+ in lepfnr·nadp+ crystal structure, thus facilitating interactions between adenine phosphate and its side chain hydroxyl group. this feature was also observed in the crystal structure of anabena fnr. nadp+ binding also causes changes in arg conformation, which also is part of the nadp+ binding site. nadp+ molecule is attached to lepfnr c-terminal domain by a network of hydrogen bonds. similarly to fad binding, the adenine portion of nadp+ is tightly bound to lepfnr. in opposite, the nicotinamide group has an increased mobility, as indicated by crystallographic b-factors. at atomic level, adenine moiety makes water-mediated hydrogen bonds with gly gly met and ile main chain atoms. the ribose group also interacts with ser and forms water-mediated hydrogen bonds with ala the phosphate group is anchored by the hydrogen bonds with arg and arg all residues mentioned above have their counterparts in the already identified crystal structures of pea and anabaena fnrs complexed with nadp+. at variance, in lepfnr the nicotinamide portion of nadp+ makes water mediated hydrogen bonds with asp arg and glu lysine also interacts to nadp+ by a hydrogen bond with oxygen of phosphate group, as indicated in figure 4b. the overall structural adjustments induced by nadp+ binding in lepfnr structure are modest, and rmsd deviations between nadp+ bound and nadp+-free states are of Å as calculated for cα positions only. structural superposition of the 3d models using their n-terminal domains shows a maximum displacement of Å between respective cα positions in these two models, which is confined to the c-terminal helices region. the superposition of the structures is given in figure enzymatic activity as shown in table lepfnr has a high diaphorase activity, reaction commonly used to characterize fnrs. similarly, lepfnr exhibited km for nadp+ which is identical to the one obtained for the pea enzyme. the kcat/km for lepfnr is circa 75% with respect to that of the latter fnr. these steady state kinetic studies reveal that the lepfnr enzyme has high affinity for nadph. the affinity for nadp+ was determined by difference absorption spectroscopy after successive addition of the nucleotide. the lepfnr enzyme has a kd for nadp+ of μm, which is in the same range of the affinities observed for the p. sativum enzyme and other reductases. these data are in agreement with the suggested affinities for nadph deduced from steady state kinetic studies. the different parameters were obtained as described in experimental procedures. each parameter value represents the average of independent experiments.  crystal structures of lepfnr reveal a typical plastidic type reductase, analogous in its folding architecture to the enzymes found in plant and cyanobacteria, in spite of displaying an important divergence at primary structure level. the typical fad and nadp+ binding domains are well conserved among them and the amino acids involved in prosthetic group and nadp+ binding have a high degree of structural conservation. our analysis reveal several distinct key features of this structure: 1) the fad molecule is bound in an extended conformation and the adenosine moiety is interacting with a different aromatic residue than in all other plastidic reductases; 2) the poorly conserved amino acid fragment 113-efiikrdn- still contains both basic amino acids that participate in the fad binding; 3) the carboxyl terminal amino acid is a tyrosine, that is planar to the isoalloxazine at Å distance. all these features of the lepfnr are found in enzymatically highly efficient reductases and are in a good agreement with the biochemical data we have obtained for this enzyme. structural comparison between present lepfnr crystal structures and the x-ray structure of the previously determined anabaena fnr complexed with nadp+ reveals a number of important similarities in the model of substrate binding between these two enzymes. the adenosine part of nadp+ and the 2'phosphate group of the substrate are in equivalent positions in these two structures. in both models the nicotinamide reactive part of substrate is not positioned in a productive form and was found too far from the isoalloxazine to allow electron transfer. the position observed for the anabaena fnr·nadp+ complex is somewhat different than the observed in lepfnr·nadp+ crystal structure. a sequence of three steps for the coenzyme recognition and binding mechanism has been proposed to explain the different conformation obtained in the anabaena fnr·nadp+ complex. here, the position adopted by the nadp+ nicotinamide bound to the lepfnr enzyme may represent another intermediate before the productive binding of the substrate. an interesting outcome of our analysis is that the binding of the substrate to holoenzyme produces not only a rearrangement of some of the residues involved in its binding but also a moderate but indubitable conformational change in the carboxyl terminal domain. this structural rearrangement can help to explain the strong negative cooperativity that has been observed for the ferredoxin and nadp+ binding. atomic coordinates and structure factors were deposited to the protein data bank and received accession codes 2rc and 2rc 
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. a number of structural features distinguish plant and bacterial fnrs, one of which is the mode of the cofactor fad binding. 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. ferredoxin-nadp reductases are flavoenzymes that catalyze the electron transfer between nadp and the proteins ferredoxin or flavodoxin. 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. leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. the comparison reveals significant structural similarities of the enzyme with the plastidic type fnrs and differences with the bacterial enzymes. leptospira interrogans fnr displays low sequence identity with plant and bacterial fnrs. the wealth of structural information about lepfnr provides a molecular basis for advanced drugs developments against leptospirosis. the crystal structures of the fad-containing lepfnr and the complex of the enzyme with nadp+, were solved and compared to known fnrs.
 ferredoxin-nadp reductases are flavoenzymes that catalyze the electron transfer between nadp 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 displays low sequence identity with plant and bacterial fnrs. however, lepfnr contains all consensus sequences that define the plastidic class fnrs.  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.  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. 