Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration

NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving (D)V values of 2.2 and 1.7, respectively, and (D)V/K(m,phosphite) values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 microm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 microm, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for (D)V and k(cat) implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH-E175A/A176R in a model system in which xylose was converted into xylitol by NADP-dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH-E175A/A176R than with either WT PTDH or a NADP-specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.     

Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration

NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T50 is 20 degrees C higher and half-life of thermal inactivation at 45 degrees C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50 degrees C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to l-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD⁺ and NADP⁺

A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air.     

Oxidation and Reduction of D-Xylose by Cell-Free Extract of Pichia quercuum

The fermentation mechanism of the simultaneous production of D-xylonic acid and xylitol from D-xylose by Pichia quercuum was studied by using a cell-free enzyme preparation. Nicotinamide adenine dinucleotide phosphate (NADP)-dependent D-xylose dehydrogenase activity and NADP-dependent D-xylose reductase activity were detected, and the oxido-reduction reaction of D-xylose was able to couple through regeneration of NADP and NADPH to produce D-xylonic acid and xylitol.     

Spore‐displayed enzyme cascade with tunable stoichiometry

Taking the advantages of inert and stable nature of endospores, we developed a biocatalysis platform for multiple enzyme immobilization on Bacillus subtilis spore surface. Among B. subtilis outer coat proteins, CotG mediated a high expression level of Clostridium thermocellum cohesin (CtCoh) with a functional display capability of ～10⁴ molecules per spore of xylose reductase‐C. thermocellum dockerin fusion protein (XR‐CtDoc). By co‐immobilization of phosphite dehydrogenase (PTDH) on spore surface via Ruminococcus flavefaciens cohesin‐dockerin modules, regeneration of NADPH was achieved. Both xylose reductase (XR) and PTDH exhibited enhanced stability upon spore surface display. More importantly, by altering the copy numbers of CtCoh and RfCoh fused with CotG, the molar ratio between immobilized enzymes was adjusted in a controllable manner. Optimization of spore‐displayed XR/PTDH stoichiometry resulted in increased yields of xylitol. In conclusion, endospore surface display presents a novel approach for enzyme cascade immobilization with improved stability and tunable stoichiometry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:383–389, 2017     

Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).     

Growth characteristics and metabolic flux analysis of Candida milleri

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.     

Potential applications of an alcohol-aldehyde/ketone oxidoreductase from thermophilic bacteria

Practical uses of a novel alcohol dehydrogenase from Thermoanaerobium brockii have been examined in crude and purified form. Stoichiometric reduction of NADP (50 mg) was demonstrated with agarose-immobilized enzyme and 0.3 (v/v) 2-propanol solution as reductant. A coenzyme recycle number of 20000 was achieved in enzymatic reactions that employed the alcohol dehydrogenase for NADPH/NADP regeneration. Gram-scale synthesis of chiral R(+) 2-pentanol was shown in a system composed of enzyme, 2-pentanone and 2-propanol as reductant. The effect of temperature, reaction time and substrate concentration on alcohol optical purity was examined. An optical purity of 80% was achieved in the enzymatic synthesis of R(+) 2-pentanol. The enzyme was easily immobilized and stable on an enzyme electrode for analytical detection of alcohols and carbonyls. T. brockii enzyme has potential applications as a commercial alcohol dehydrogenase because of broad substrate specificity and activity at high temperature or high solvent concentration, rare carbonyl si-face stereo-specificity in hydrogen transfer, and high stability and activation of immobilized enzyme.     

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.     

Cancer-associated isocitrate dehydrogenase mutations inactivate NADPH-dependent reductive carboxylation

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP(+)-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO(2)-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP(+) and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired.     

Pre-steady-state studies of phosphite dehydrogenase demonstrate that hydride transfer is fully rate limiting

Phosphite dehydrogenase (PTDH) is a unique NAD-dependent enzyme that catalyzes the oxidation of inorganic phosphite to phosphate. The enzyme has great potential for cofactor regeneration, and mechanistic studies have provided some insight into the residues that are important for catalysis. In this investigation, pre-steady-state studies were performed on the His6-tagged wild-type (WT) enzyme, several active site mutants, a thermostable mutant (12X-PTDH), and a thermostable mutant with dual cofactor specificity (NADP-12X-PTDH). Stopped-flow kinetic experiments indicate that slow steps after hydride transfer do not significantly limit the rate of reaction for the WT enzyme, the active site mutants, or the thermostable mutant. Pre-steady-state kinetic isotope effects (KIEs) and single-turnover experiments further confirm that slow steps after the chemical step do not significantly limit the rate of reaction for any of these proteins. Collectively, these results suggest that the hydride transfer step is fully rate determining in PTDH and that the observed KIE on kcat is the intrinsic effect in WT PTDH and the mutants examined. In contrast, a slow step after catalysis may partially limit the rate of phosphite oxidation by NADP-12X-PTDH with NADP as the cofactor. Finally, site-directed mutagenesis of Asp79 indicates that this residue is important in orienting Arg237 for proper interaction with phosphite.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

Evidence for the stabilization of NADPH relative to NADP(+) on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence.

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria (hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase (rrdIII.D155W), are more fluorescent when NADP(+) is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP(+)/NADPH bound to the dIII proteins is some 60-70 mV higher than that in free solution. This results in favoured reduction of NADP(+) by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.     

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.     

Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.     

Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens

The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD(+) kinase (NADK) and NADP(+) phosphatase (NADPase), enzymes known to regulate the levels of NAD(+) and NADP(+). While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.     

Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase

Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in beta-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP(+)-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP(+)-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP(+)-linked acetaldehyde dehydrogenase and NADP(+)-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and beta-lactam production and may provide an interesting target for metabolic engineering.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration

The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD(+) (1.7 ? resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 ? resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.