Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus.
ABSTRACT: Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5?Å resolution from crystals belonging to the orthorhombic space group P2?2?2, with unit-cell parameters a=57.2, b=164.3, c=95.0?Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.
Project description:Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP(+) domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.
Project description: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.
Project description:The flavodoxins constitute a highly conserved family of small, acidic electron transfer proteins with flavin mononucleotide prosthetic groups. They are found in prokaryotes and in red and green algae, where they provide electrons at low potentials for the reduction of nitrogen by nitrogenase, for the light-dependent reduction of NADP+ in photosynthesis, and for the reduction of sulfite. Proteins with the physical characteristics of flavodoxins have been implicated in the reductive activation of pyruvate formate-lyase and cobalamin-dependent methionine synthase in Escherichia coli. We have purified flavodoxin to homogeneity from E. coli, determined its N-terminal amino acid sequence, and used this sequence to construct a 64-fold degenerate oligonucleotide probe for the flavodoxin gene. Because the phenotype of a flavodoxin mutant is not known, we used this degenerate probe to screen the phages of the Kohara library and identified two phages, with inserts mapping at approximately 16 min, that hybridized to the probe. The flavodoxin gene, designated fldA, was subcloned from the DNA in the overlap region of these two clones. The deduced amino acid sequence, determined by nucleotide sequencing of the flavodoxin gene, shows strong homology with flavodoxins from nitrogen-fixing bacteria and cyanobacteria. The fldA gene maps at 15.9 min on the E. coli chromosome and is transcribed in a counterclockwise direction.
Project description:Low-molecular-weight (low M<sub>r</sub> ) thioredoxin reductases (TrxRs) are homodimeric NADPH-dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx-like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR-like ferredoxin/flavodoxin NADP<sup>+</sup> oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant-type FNR. In Bacillus cereus, a TrxR-like FNR has been shown to reduce the flavodoxin-like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR and lack the homologous TrxR-like FNR. Due to the structural similarity between TrxRs and TrxR-like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low M<sub>r</sub> TrxR may be able to replace TrxR-like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity toward NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co-substrate binding in TrxR.
Project description:1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.
Project description:Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 66.49, c = 121.32 A. X-ray data sets have been collected to 2.17 A resolution.
Project description:Bacillus subtilis yumC encodes a novel type of ferredoxin-NADP+ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP+ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH-thioredoxin reductase (TdR) and TdR-like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP+ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C-terminal extension, not found in E. coli TdR but in TdR-like protein from T. thermophilus HB8, covers the re-face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD-binding region and His324 in the C-terminal extension stack on the si- and re-faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid-type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid-type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2'-phosphate of NADP+, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re-face of FAD is hardly relevant to the nucleotide selectivity.
Project description:Ferredoxin (Fd) interacts with ferredoxin-NADP(+) reductase (FNR) to transfer two electrons to the latter, one by one, which will finally be used to reduce NADP(+) to NADPH. The formation of a transient complex between Fd and FNR is required for the electron transfer (ET), and extensive mutational and crystallographic studies have been reported to characterize such protein-protein interaction. However, some aspects of the association mechanism still remain unclear. Moreover, in spite of their structural differences, flavodoxin (Fld) can replace Fd in its function and interact with FNR to transfer electrons with only slightly lower efficiency. Although crystallographic structures for the FNR:Fd association have been reported, experimental structural data for the FNR:Fld interaction are highly elusive. We have modeled here the interactions between FNR and both of its protein partners, Fd and Fld, using surface energy analysis, computational rigid-body docking simulations, and interface side-chain refinement. The results, consistent with previous experimental data, suggest the existence of alternative binding modes in these ET proteins.
Project description:Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.
Project description:Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.