NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

A chimeric bifunctional enzyme composing of galactose dehydrogenase (galDH; from Pseudomonas fluorescens) and lactate dehydrogenase (LDH; from Bacillus stearothermophilus) was successfully constructed. The chimeric galDH/LDH possessed dual characteristics of both galactose dehydrogenase and lactate dehydrogenase activities while exhibiting hexameric rearrangement with a molecular weight of approximately 400 kDa. In vitro observations showed that the chimeric enzyme was able to recycle NAD with a continuous production of lactate without any externally added NADH. Two fold higher recycling rate (0.3 mM/h) than that of the native enzyme was observed at pH values above 8.5. Proximity effects became especially pronounced during the recycling assay when diffusion hindrance was induced by polyethylene glycol. All these findings open up a high feasibility to apply the NAD(H) recycling system for metabolic engineering purposes e.g. as a model to gain a better understanding on the molecular proximity process and as the routes for synthesizing of numerous high-value-added compounds.     

Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast.

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.     

Amino acid composition and N-terminal sequence of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

Abstract The NADP-linked glutamate dehydrogenase (NADP-GDH) from epimastigotes of Trypanosoma cruzi , Tul 2 stock, has been purified by an improved procedure. The enzyme has subunit molecular weight (47 kDa), amino acid composition and N-terminal sequence similar to those of the NADP-GDH from Escherichia coli , including the N-terminal extension of 15 amino acids present in the E. coli enzyme, but not in the NADP-GDH from Neurospora crassa .     

Effect of acid trehalase (ATH) on impaired yeast vacuolar activity

In this study, this protein was overexpressed in yeast cells grown on trehalose-containing medium to assess its impact on yeast vacuolar activity. ATH was confirmed to be located in both cell surface and vacuoles and the overexpression of ATH was observed to decrease vacuolar activity. Therefore, an assumption was suggested to explain this phenomenon as follows: when grown on containing trehalose medium, the ATH localization at cellular periplasm, but not the vacuole, is prioritized to utilize the extracellular trehalose for cell growth. The multivesicular body pathway (MVB pathway) via which ATH is transported into vacuoles is believed to be down-regulated to favor the accumulation of ATH at cell surface area. By extension, other vacuolar proteins travelling through MVB pathway to reach yeast vacuoles likely also suffer the down regulation. It can be concluded that acid trehalase may contribute down regulation of other vacuolar proteins through MVB pathway. This study suggests that it is a potential of acid trehalase (ATH) on impaired activity of yeast vacuolar.     

Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.     

The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase

Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.     

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)⁺-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO⁻ aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd²⁺, Co²⁺, Hg²⁺, Mg²⁺, Pb²⁺ and Zn²⁺ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD⁺-GDH activity was negligible. Among the applied ions, only Ca²⁺ activated NADH-GDH.     

Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus

In Picrophilus torridus, a euryarchaeon that grows optimally at 60 degrees C and pH 0.7 and thus represents the most acidophilic thermophile known, glucose oxidation is the first proposed step of glucose catabolism via a nonphosphorylated variant of the Entner-Doudoroff pathway, as deduced from the recently completed genome sequence of this organism. The P. torridus gene for a glucose dehydrogenase was cloned and expressed in Escherichia coli, and the recombinant enzyme, GdhA, was purified and characterized. Based on its substrate and coenzyme specificity, physicochemical characteristics, and mobility during native PAGE, GdhA apparently resembles the main glucose dehydrogenase activity present in the crude extract of P. torridus DSM 9790 cells. The glucose dehydrogenase was partially purified from P. torridus cells and identified by MS to be identical with the recombinant GdhA. P. torridus GdhA preferred NADP+ over NAD+ as the coenzyme, but was nonspecific for the configuration at C-4 of the sugar substrate, oxidizing both glucose and its epimer galactose (Km values 10.0 and 4.5 mM, respectively). Detection of a dual-specific glucose/galactose dehydrogenase points to the possibility that a 'promiscuous' Entner-Doudoroff pathway may operate in P. torridus, similar to the one recently postulated for the crenarchaeon Sulfolobus solfataricus. Based on Zn2+ supplementation and chelation experiments, the P. torridus GdhA appears to contain structurally important zinc, and conserved metal-binding residues suggest that the enzyme also contains a zinc ion near the catalytic site, similar to the glucose dehydrogenase enzymes from yeast and Thermoplasma acidophilum. Strikingly, NADPH, one of the products of the GdhA reaction, is unstable under the conditions thought to prevail in Picrophilus cells, which have been reported to maintain the lowest cytoplasmic pH known (pH 4.6). At the optimum growth temperature for P. torridus, 60 degrees C, the half-life of NADPH at pH 4.6 was merely 2.4 min, and only 1.7 min at 65 degrees C (maximum growth temperature). This finding suggests a rapid turnover of NADPH in Picrophilus.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.