Malate Dehydrogenase, Alcohol Dehydrogenase, and 6-Phosphogluconate Dehydrogenase Isozymes of Zea mays L. × Tripsacum dactyloides L. Hybrids and Parents

Electrophoretic patterns of malate dehydrogenase (Mdh), alcohol dehydrogenase (Adh), and 6-phosphogluconate dehydrogenase (Pgd) of Zea mays L. × Tripsacum dactyloides L. hybrids and their parents were compared. The components of enzymes specific to T. dactyloides may be used as markers to identify the following T. dactyloides chromosomes in the hybrids: Tr 16 (Mdh 2 and Pdg 1), Tr 7, and/or Tr 13 (Adh 2). The isozymes of Mdh 2 are supposed as a possible biochemical marker to evaluate the introgression of genes, determining an apomictic mode of reproduction from T. dactyloides (localized on Tripsacum 16 chromosome) into Z. mays. The isozymes may be used as markers for the identification of maize chromosomes 1 and 6 in the hybrids as well. Chromosome count taken on the examined hybrids showed the addition of 9 to 13 chromosomes of T. dactyloides to maize chromosome complement.     

Production,Purification and Crystallization of 3α-Hydroxysteroid Dehydrogenase of Pseudomonas putida

The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B–11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and column chromatographies on DEAE-celluIose, Sephadex G–100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (s_20,w=3.2).     

Ontogenesis of Oxidative Reaction of 17β-hydroxysteroid Dehydrogenase and 11β-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study

The enzyme 17β-hydroxysteroid dehydrogenase is required for the synthesis and 11β-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17β-hydroxysteroid dehydrogenase (substrate: 5-androstene-3β, 17β-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17β-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11β-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17β-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11β -hydroxysteroid dehydrogenase in addition to types 1 and 2.     

3α-Hydroxysteroid Dehydrogenase in Animal and Human Tissues

This review analyzes data on the biological role of 3-hydroxysteroid dehydrogenase (3-HSD) in animal and human tissues and describes its main characteristics, mechanism of action, and regulation of activity. Based on published data, a scheme for the actions of androgen, progestin, and glucocorticoids involving the participation of 3-HSD is proposed. According to this scheme, in the mechanism of steroid action 3-HSD not only regulates the concentration of the main effector androgen, 5-dihydrotestosterone, in target cells, but also switches androgen, progestin, and glucocorticosteroid genomic activity to non-genomic activity.     

NAD-Dependent Maltose Dehydrogenase and NAD-Dependent d-Glucose Dehydrogenase of Alkalophilic Corynebacterium sp. No. 150–1

An alkalophilic bacterium, strain No. 150–1, which had NAD-dependent sugar dehydrogenase activities on maltose (NAD-MalDH) and d-glucose (NAD-GlcDH), was isolated from soil. This microorganism was identificd to be in a strain of the genus Corynebacterium. The bacterium grew to similar degrees at Na₂CO₃ concentrations from 0 to 0.5%. NAD-MalDH and NAD-GlcDH were not inducible types. Soybean casein was the most effective nitrogen source for enzyme production. Activity staining of these two dehydrogense on polyacrylamide gel showed that these activities were derived from two different proteins. The cell free extract did not contain NADP-dependent maltose dehydrogenase.     

Methodology and Problems of Protein‐Ligand Docking: Case Study of Dihydroorotate Dehydrogenase,Thymidine Kinase,and Phosphodiesterase 4

Abstract

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear‐cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.     

Molecular Framework of Steroid/Retinoid Discrimination in 17β-Hydroxysteroid Dehydrogenase Type 1 and Photoreceptor-associated Retinol Dehydrogenase

Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17β-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17β-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17β-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17β-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17β-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17β-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17β-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17β-HSD1 developed for cancer treatment.     

Presence and Regulation of α-Ketoglutarate Dehydrogenase Complex in a Glutamate-Producing Bacterium,Brevibacterium flavum

The presence of α-ketoglutarate (α-KG) dehydrogenase complex in the glutamate-producing bacteria was demonstrated for the first time with Brevibacterium flavum. The partially purified enzyme, which was specific to KG and NAD⁺ with the usual requirements for other co-factors, was labile and stabilized by glycerol, Mg²⁺, and thiamine pyrophosphate. The enzyme showed an optimum pH of 7.6 and K_ms of 80, 86, and 61 μm for KG, NAD⁺, and CoA, respectively, cis-Aconitate, succinyl-CoA, NADPH, NADH, pyruvate, and oxalacetate strongly inhibited the activity, while it was activated by acetyl-CoA, but not by AMP. Various inorganic and organic salts also inhibited the activity. When cells were cultured in glucose and acetate media, the specific activity of the cell extracts increased markedly and reached to a maximum at the late-logarithmic phase. Then, it decreased to the basal level. The addition of glutamate stimulated the synthesis of the enzyme.     

Human γ-Aminobutyraldehyde Dehydrogenase (ALDH9): cDNA Sequence, Genomic Organization, Polymorphism, Chromosomal Localization, and Tissue Expression

The cDNA and the gene (ALDH9) for a human aldehyde dehydrogenase isozyme, which has a high activity for oxidation of γ-aminobutyraldehyde and other amino aldehydes, were cloned and characterized. The cDNA has an open reading frame of 1479 bp encoding 493 amino acid residues. The gene is about 45 kb and consists of 10 coding exons interrupted by nine introns. The gene was assigned to chromosome 1q22–q23, using fluorescencein situhybridization. Northern blot hybridization indicated that the size of the mRNA is about 2.4 kb and that the gene is expressed at high levels in adult liver, skeletal muscle, and kidney and low levels in heart, pancreas, lung, and brain. The gene is polymorphic, i.e., C or T at nt 327 and C or G at nt 344.     

Dihydroorotate Dehydrogenase mRNA and Protein Expression Analysis in Normal and Drug‐Resistant Cells

To follow the expression of the fourth enzyme of pyrimidine de novo synthesis dihydroorotate dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT‐PCR in rat tissues. Rabbit polyclonal anti‐DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B‐lymphocytes, which were adapted to tolerate up to a 50‐fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

α-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH₄) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. ¹³C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.All aerobic organisms have to contend with the dangers associated with reactive oxygen species (ROS), toxic moieties that are routinely generated as a consequence of ATP production via oxidative phosphorylation (34). The transfer of electrons from NADH and reduced flavin adenine dinucleotide to oxygen is mediated by the respiratory complexes, the major sites of intracellular ROS generation (1). These by-products of oxidative phosphorylation are very harmful and have to be nullified if organisms are to survive in an aerobic environment (24). If left unchecked, ROS can damage biological macromolecules, leading to the demise of the cell. Hence, it is not surprising that all aerobic organisms have devised intricate antioxidative defense strategies in an effort to proliferate in the presence of oxygen.Enzymes such as superoxide dismutase and catalase are uniquely bestowed with the task of eliminating superoxide and hydrogen peroxide, two important ROS (11, 12, 21). Glutathione (GSH), a tripeptide, also plays a pivotal role in the detoxification of ROS (22). However, to be effective, all these ROS disposal processes have to be regenerated with the aid of NADPH. This nicotinamide dinucleotide is the main power behind all antioxidative defense strategies, as it provides the reducing fuel necessary to recharge all effectors involved in combating ROS (32). Thus, various enzyme systems and metabolic networks that orchestrate the biogenesis of NADPH have to be activated if an organism is to acquire ATP via the reduction of oxygen (23). We have recently shown the crucial role played by NADK, an enzyme that mediates the formation of NADP, a key ingredient known to tilt cellular metabolism toward the synthesis of NADPH and away from the formation of NADH, a prooxidant (28). Hence, aerobic respiration, ROS production, and antioxidative defense strategies have to be intricately modulated.Although the elimination of ROS is critical to the survival of all organisms, it is also important to appreciate the role that adaptative mechanisms play to lower the production of ROS. Our laboratory has recently identified an intriguing role of the tricarboxylic acid (TCA) cycle in this regard (15, 27). By modulating the production of NADH and NADPH, this metabolic network appears to be instrumental in striking the proper balance between the generation of ROS and the aerobic formation of ATP. As part of our study to delineate the molecular mechanisms that allow cellular systems to adapt to oxidative stress, we have identified how α-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) play a critical role in modulating α-ketoglutarate (KG) homeostasis in Pseudomonas fluorescens challenged with menadione. This keto acid can readily nullify these ROS with the concomitant formation of succinate, a moiety that may signal anaerobic metabolism. The roles of KGDH in sensing ROS and limiting NADH production are also discussed.     

The Isolation,Purification, and Some Properties of NAD-Dependent Isocitrate Dehydrogenase from the Organic Acid–Producing Yeast Yarrowia lipolytica

The NAD⁺-dependent isocitrate dehydrogenase of the organic acid–producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The K _m for NAD⁺ is 136 M, and that for isocitrate is 581 M. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD⁺) in the overproduction of citric and keto acids is discussed.     

Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid β and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production

We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease.     

DSD – An integrated,web-accessible database of Dehydrogenase Enzyme Stereospecificities

Background  

Dehydrogenase enzymes belong to the oxidoreductase class and utilise the coenzymes NAD and NADP. Stereo-selectivity is focused on the C4 hydrogen atoms of the nicotinamide ring of NAD(P). Depending upon which hydrogen is transferred at the C4 location, the enzyme is designated as A or B stereospecific.     

Subcellular Localization and Properties of L-Galactono-γ-lactone Dehydrogenase in Spinach Leaves

L-Galactono-γ-lactone dehydrogenase, which catalyzes the final step of the biosynthesis of L-ascorbate, is bound to spinach mitochondrial membrane, as confirmed by linear sucrose density gradient centrifugation. The solubilized enzyme was very labile, but stabilized in the presence of L-galactono-γ-lactone under anaerobic conditions. The enzyme reduced cytochrome c and phenazine methosulfate in the presence of L-galactono-γ-lactone, but not when L-gulono-γ-lactone was used as an electron donor. The K_ms of the enzyme for L-galactono-γ-lactone and cytochrome c were 192 μM and 180 μM, respectively.     

Circadian Variations in the Activities of 6‐Phosphogluconate Dehydrogenase and Glucose‐6‐Phosphate Dehydrogenase in the Liver of Conrol and Streptozotocin‐Induced Diabetic Rats

The aim of this study was to examine: the 24 h variation of 6‐phosphogluconate dehydrogenase and glucose‐6‐phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin‐induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light‐dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6‐phosphogluconate dehydrogenase activity was measured by substituting 6‐phosphogluconate as substrate. Glucose‐6‐phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two‐way ANOVA revealed that 6‐phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6‐phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose‐6‐phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6‐phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one‐way ANOVA). Time‐dependent variation in glucose‐6‐phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ‐treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH‐generating enzymes exhibit 24 h variation, which is not influenced by diabetes.