Enzymic and Structural Studies on Drosophila Alcohol Dehydrogenase and Other Short-Chain Dehydrogenases/Reductases

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters k_cat and K_m. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(−) alcohols. At a high concentration of R(−) alcohols substrate activation occurs. The k_cat and K_m values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD⁺. Probably these lyases descend from an SDR, which has lost the capability to bind NAD⁺, but the enzyme reaction mechanisms may still be similar. Received: 23 May 2000 / Accepted: 4 January 2001     

Comparative analysis of two members of the metal ion-containing group III-alcohol dehydrogenases from Dickeya zeae

Purpose of work

A pair of NAD⁺- and NADP⁺-dependent group III-alcohol dehydrogenases was characterized from the enterobacterium, Dickeya zeae, to expand our understanding of the distribution and biochemical properties of this interesting group of enzymes. Two putative group III-alcohol dehydrogenases (ADHs) were identified in the genome of Dickeya zeae. Amino acid alignments and phylogenetic analysis revealed that Adh3.1 and Adh3.2 are only distantly related (~25 % identity at the protein level). Both proteins were purified to homogeneity after heterologous expression in E. coli. A specific activity of 1.8 U/mg was measured for the NAD⁺-dependent enzyme Adh3.1 with ethanol used as substrate, while NADPH-dependent Adh3.2 preferred butanal (29.1 U/mg) as substrate. Maximum activity for Adh3.1 was at 50 °C and pH 10 and for Adh3.2 at 70 °C and pH 6. Cell viability assays were used to confirm activity towards butanal and glyoxals. Biochemical characterization and phylogenetic analyses led to the hypothesis that Adh3.1 and Adh3.2 are probably the result of an ancient gene duplication event followed by functional diversification.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation

Malic enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO₂ providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD⁺ as the primary cofactor. K_m values for NAD⁺ and NADP⁺ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP⁺- but not NAD⁺-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP⁺-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.     

The interactions of artificial coenzymes with alcohol dehydrogenase and other NAD(P)(H) dependent enzymes

The interactions of CL4, a biomimetic analogue of NAD⁺ comprising a nicotinamide functionality coupled via a triazine ring to a dibenzenesulphonic acid unit, and of a series of analogues, with HLADH and other dehydrogenases have been compared to those of the natural coenzymes NAD(P)⁺. CL4, together with one analogue with one of the sulphonic acid groups shifted by one position and another analogue with a single benzenedisulphonic acid unit, have been shown to be functional mimics of NAD⁺ in the oxidation of butan-1-ol by horse liver alcohol dehydrogenase (HLADH). A combination of discontinuous HPLC-based assays and continuous fluorescence based assays were used to deduce approximate kinetic constants for this reaction, using the artificial coenzymes, at pH 7.5 and 37°C. HLADH demonstrated a V_max with the most active analogue which was 4% of that with NAD⁺. The substrate specificity of HLADH using these coenzymes was found to change relative to that using the natural coenzyme. Activity was sought from a range of other dehydrogenases: Bacillus megaterium glucose dehydrogenase, Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and sheep liver sorbitol dehydrogenase; all displayed activity using a range of the biomimetic coenzymes.     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

     

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase–ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E·NAD⁺) and three ternary (E·NAD⁺·acetone, E·NAD⁺·3-pentanone and E·NAD⁺·cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

l-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. l-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP⁺-dependent enzymes from chloroplasts and was separated from the NAD⁺-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis–Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K _m for oxaloacetate was 20 μM; the K _m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD⁺ increased with pH but there was very little activity with NADP⁺. At pH 7.0, the K _m for l-malate was 5 mM and the K _m for NAD⁺ was 24 μM. The reductive activity was quite insensitive to inhibition by l-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10–20 times higher than the oxidative activity. These results indicate that the l-malate dehydrogenase in N. europaea is similar to other NAD⁺-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.     

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Biocatalysts that mediate the H₂-dependent reduction of NAD⁺ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD⁺-reducing [NiFe]?hydrogenase that sustains catalytic activity at high temperatures and in the presence of O₂, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD⁺-reducing [NiFe]?hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1^T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H₂-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H₂-mediated NAD⁺ reduction activity was observed at 80 °C and pH 6.5, and catalytic activity was found to be sustained at low O₂ concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]?hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD⁺-reducing [NiFe]?hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H₂-driven cofactor recycling under oxic conditions at elevated temperatures.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

Studies on the metabolism of galactose-6-phosphate in rat heart and brain.

The subcellular distribution of NADP⁺ and NAD⁺-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP⁺ or NAD⁺ as cofactor. While all of the tissues examined had NADP⁺-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP⁺-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (K_m's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP⁺ or NAD⁺ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (K_i = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.     

Design,synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Nicotinamide adenine dinucleotide (NAD⁺) synthetase catalyzes the last step in NAD⁺ biosynthesis. Depletion of NAD⁺ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD⁺ synthetase (NadE) from Mtb, we expect to eliminate NAD⁺ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC₅₀ = 1000 µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC₅₀ value of 90 µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100 µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.