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.

Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (<10 s(-1)). Mixing BsFd(red) with BsFNR(ox) induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs.

The role of the highly conserved C266 and L268 of pea ferredoxin-NADP(+) reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP(+) reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268-270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).

Cytoplasmic NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) has been purified and characterized, and its gene sequenced in many animal, plant, and yeast species. However, much less information is available on this enzyme-gene in insects. As a first step in investigating the biochemical and molecular mechanisms by which NADP(+)-IDH contributes to adaptations for flight vs. reproduction in insects, the enzyme was purified to homogeneity in the wing-dimorphic cricket, Gryllus firmus, characterized, and its corresponding gene sequenced. Using a combination of polyethylene glycol precipitation, Cibacron-Blue affinity chromatography, and hydrophobic interaction chromatography the enzyme was purified 291-fold (7% yield; specific activity = 15.8 µmol NADPH/min/mg protein). The purified enzyme exhibited a single band on SDS PAGE (46.3 kD), but consisted of two N-terminal amino acid sequences that differed in the first two amino acids. Purified enzyme exhibited standard Michaelis-Menten kinetics at pH 8.0 and 28° C (K(M(NADP+)) = 2.3 ± 0.4 µM; K(M(Na+-Isocitrate)) = 14.7 + 1.8 µM). Subunit molecular mass and K(M)S were similar to published values for NADP(+)-IDHs from a variety of vertebrate and two insect species. PCR amplification of an internal sequence using genomic DNA followed by 3' and 5' RACE yielded a nucleotide sequence of the mature protein and translated amino-acid sequences that exhibited high similarity (40-50% and 70-80%, respectively) to sequences from insect and vertebrate NADP(+)-IDHs. Two potential ATG start codons were identified. Both Nterminal amino-acid sequences matched the nucleotide sequence, consistent with both enzyme forms being transcribed from the same gene, although these variants could also be encoded by different genes. Bioinformatic analyses and differential centrifugation indicated that the majority, if not all, of the enzyme is cytoplasmic. The enzyme exhibited high specific activity in fat body, head and gut, and a single band on native PAGE.

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 μM and ∆H of -20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.

NADP-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-H4MPT to form methenyl-H4MPT+ by using NADP+ as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO2 in methylotrophic and methanotrophic bacteria. Here, we report on the crystal structures of the ternary MtdA-substrate complexes from Methylorubrum extorquens AM1 obtained in open and closed forms. Their conversion is accomplished by opening/closing the active site cleft via a 15° rotation of the NADP, relative to the pterin domain. The 1.08 Å structure of the closed and active enzyme-NADP-methylene-H4MPT complex allows a detailed geometric analysis of the bulky substrates and a precise prediction of the hydride trajectory. Upon domain closure, the bulky substrate rings become compressed resulting in a tilt of the imidazolidine group of methylene-H4MPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP+ and methenyl-H4MPT+ revealed an extremely short distance between nicotinamide-C4 and imidazoline-C14a of 2.5 Å, which demonstrates the strong pressure imposed. The pterin-imidazolidine-phenyl butterfly angle of methylene-H4MPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H2- and F420-dependent methylene-H4MPT dehydrogenases. The concept of compression-driven hydride transfer including quantum mechanical hydrogen tunneling effects, which are established for flavin- and NADP-dependent enzymes, can be expanded to hydride-transferring H4MPT-dependent enzymes.

An NAD(+)-specific glutamate dehydrogenase (NAD-GDH) that is inducible by L-glutamine was isolated from Achlya klebsiana and purified to electrophoretic homogeneity. The enzyme is only partially active in vitro unless NADP+ (an activator) is present in both its oxidative deamination and reductive amination reactions. This type of enzyme was reported (LéJohn, H.B. (1971) Nature 231, 164-168) to be widespread among the amorphous group of algae-related fungi classified as Oomycota. The enzyme retained its dependence on NADP+ at all stages of its purification. NADP+ decreased the Km of substrates 3-fold and increased the Vmax 4-fold. M(r) of the undernatured enzyme was 480,000, and, denatured, only a single subunit of M(r) 120,000 was seen. A polyclonal antibody raised in rabbit against purified enzyme subunit excised from SDS-polyacrylamide gel electrophoresis gels immunoprecipitated the M(r) 120,000 polypeptide, the undenatured enzyme, and a physically distinct polypeptide of M(r) 74,000. The antibody, purified against the M(r) 120,000 enzyme subunit as anchored antigen on Sepharose, still immunoprecipitated the M(r) 74,000 polypeptide. The M(r) 74,000 polypeptide was found to be a subunit of a M(r) 220,000 native protein.

Trypanosoma cruzi exhibits two putative isocitrate dehydrogenases (IDHs). Both idh genes were cloned and the recombinant enzymes expressed in Escherichia coli. Our results showed that T. cruzi IDHs are strictly dependent on NADP(+) and display apparent affinities towards isocitrate and the coenzyme in the low micromolar range. In T. cruzi, IDHs are cytosolic and mitochondrial enzymes, and there is no evidence for the typical Krebs cycle-related NAD-dependent IDH. Hence, like in Trypanosoma brucei, the Krebs cycle is not a canonical route in T. cruzi. However, the citrate produced in the mitochondrion could be isomerized into isocitrate in the cytosol and the mitochondrion by means of the putative aconitase, which would provide the substrate for both IDHs. The cytosolic IDH is significantly more abundant in amastigotes, cell-derived and metacyclic trypomastigotes than in epimastigotes. This observation fits in well with the expected oxidative burst this pathogen has to face when infecting the mammalian host.

Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH.

Cyclic electron transfer (CET) predominates when NADP+ is at basal levels, early in photosynthetic induction; however, the mechanism underlying the subsequent supply of NADP+ to fully drive steady-state linear electron transfer remains unclear. Here, we investigated whether CET is involved in de novo NADP+ supply in Arabidopsis thaliana and measured chloroplastic NADP dynamics to evaluate responsiveness to variable light, photochemical inhibitors, darkness, and CET activity. The sum of oxidized and reduced forms shows that levels of NADP and NAD increase and decrease, respectively, in response to light; levels of NADP and NAD decrease and increase in the dark, respectively. Moreover, consistent with the pH change in the stroma, the pH preference of chloroplast NAD+ phosphorylation and NADP+ dephosphorylation is alkaline and weakly acidic, respectively. Furthermore, CET is correlated with upregulation of light-responsive NADP level increases and downregulation of dark-responsive NADP level reductions. These findings are consistent with CET helping to regulate NADP pool size via stromal pH regulation under fluctuating light conditions.

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

Cofactor F420 is a 5'-deazaflavin derivative first discovered in methanogenic archaea but later found also to be present in some bacteria. As a coenzyme, it is involved in hydride transfer reactions and as a prosthetic group in the DNA photolyase reaction. We report here for the first time on the crystal structure of an F420-dependent oxidoreductase bound with F420. The structure of F420H2:NADP+ oxidoreductase resolved to 1.65 A contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 A, which is optimal for hydride transfer. Comparison of the structures bound with and without substrates reveals that of the two substrates NADP has to bind first, the binding being associated with an induced fit.

Compared to the large number of studies focused on the factors controlling C3 photosynthesis efficiency, there are relatively fewer studies of the factors controlling photosynthetic efficiency in C4 leaves. Here, we used a dynamic systems model of C4 photosynthesis based on maize (Zea mays) to identify features associated with high photosynthetic efficiency in NADP-malic enzyme (NADP-ME) type C4 photosynthesis. We found that two additional factors related to coordination between C4 shuttle metabolism and C3 metabolism are required for efficient C4 photosynthesis: (1) accumulating a high concentration of phosphoenolpyruvate through maintaining a large PGA concentration in the mesophyll cell chloroplast and (2) maintaining a suitable oxidized status in bundle sheath cell chloroplasts. These identified mechanisms are in line with the current cellular location of enzymes/proteins involved in the starch synthesis, the Calvin-Benson cycle and photosystem II of NADP-ME type C4 photosynthesis. These findings suggested potential strategies for improving C4 photosynthesis and engineering C4 rice.

The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usually specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity toward NADP+, and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60-85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s-1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.

The disulfide bond is unusual in natural products and critical for thermal stability, cell permeability and bioactivity. DepH from Chromobacterium violaceum No. 968 is an FAD-dependent enzyme responsible for catalyzing the disulfide bond formation of FK228, an anticancer prodrug approved for the treatment of cutaneous T-cell lymphoma. Here we report the crystal structures of DepH and DepH complexed with a substrate analogue S,S'-dimethyl FK228 at 1.82 Å and 2.00 Å, respectively. Structural and biochemical analyses revealed that DepH, in contrast to the well characterized low molecular weight thioredoxin reductases (LMW TrxRs), is an NADP(+)-independent dithiol oxidase. DepH not only lacks a conserved GGGDXAXE motif necessary for NADP(+) binding in the canonical LMW TrxRs, but also contains a 11-residue sequence which physically impedes the binding of NADP(+). These observations explain the difference between NADP(+)-independent small molecule dithiol oxidases and NADP(+)-dependent thioredoxin reductases and provide insights for understanding the catalytic mechanism of dithiol oxidases involved in natural product biosynthesis.

The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

YqhD, an E. coli alcohol/aldehyde oxidoreductase, is an enzyme able to produce valuable bio-renewable fuels and fine chemicals from a broad range of starting materials. Herein, we report the first computational solution-phase structure-dynamics analysis of YqhD, shedding light on the effect of oxidized and reduced NADP/H cofactor binding on the conformational dynamics of the biocatalyst using molecular dynamics (MD) simulations. The cofactor oxidation states mainly influence the interdomain cleft region conformations of the YqhD monomers, involved in intricate cofactor binding and release. The ensemble of NADPH-bound monomers has a narrower average interdomain space resulting in more hydrogen bonds and rigid cofactor binding. NADP-bound YqhD fluctuates between open and closed conformations, while it was observed that NADPH-bound YqhD had slower opening/closing dynamics of the cofactor-binding cleft. In the light of enzyme kinetics and structural data, simulation findings have led us to postulate that the frequently sampled open conformation of the cofactor binding cleft with NADP leads to the more facile release of NADP while increased closed conformation sampling during NADPH binding enhances cofactor binding affinity and the aldehyde reductase activity of the enzyme.

Sleep behavior is conserved throughout evolution, and sleep disturbances are a frequent comorbidity of neuropsychiatric disorders. However, the molecular basis underlying sleep dysfunctions in neurological diseases remains elusive. Using a model for neurodevelopmental disorders (NDDs), the Drosophila Cytoplasmic FMR1 interacting protein haploinsufficiency (Cyfip85.1/+), we identify a mechanism modulating sleep homeostasis. We show that increased activity of the sterol regulatory element-binding protein (SREBP) in Cyfip85.1/+ flies induces an increase in the transcription of wakefulness-associated genes, such as the malic enzyme (Men), causing a disturbance in the daily NADP+/NADPH ratio oscillations and reducing sleep pressure at the night-time onset. Reduction in SREBP or Men activity in Cyfip85.1/+ flies enhances the NADP+/NADPH ratio and rescues the sleep deficits, indicating that SREBP and Men are causative for the sleep deficits in Cyfip heterozygous flies. This work suggests modulation of the SREBP metabolic axis as a new avenue worth exploring for its therapeutic potential in sleep disorders.