Purification and properties of a heat-stable and cold-labile NAD-specific glutamate dehydrogenase from Sporosarcina ureae

NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (M _r 250,000±25,000) is composed of subunits identical in molecular mass (M _r 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.Abbreviation NAD-GluDH nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Purification and characterization of the NADP-dependent glutamate dehydrogenase from Bacillus fastidiosus

Ammonia assimilation in Bacillus fastidiosus proceeds via the NADP-dependent glutamate dehydrogenase. The enzyme, purified to homogeneity, is composed of identical subunits with a molecular weight of about 48 000 dalton. Presumably the enzyme is a hexamer. The enzyme is specific for NADP (H). The pH optima for the amination and deamination reactions are 7.7 and 8.6, respectively. The temperature optimum is 60°C. Furthermore, temperature stability and apparent K_m values for substrates of both the amination and deamination reactions were determined. Several metabolites were tested for their effect on the enzyme activity. Only malate and fumarate showed some inhibitory effect.Abbreviation GDH glutamate dehydrogenase     

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.     

Purification and genetic control of NAD-dependent glutamate dehydrogenase from Drosophila melanogaster

Glutamate dehydrogenase has been purified to near-homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 347,000 measured by sucrose gradient sedimentation and 343,000 measured by variable-porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions showed that the enzyme consists of six subunits of molecular weight 57,000. The structural gene for GDH has been mapped at 81.7±0.8 on the third chromosome by means of an electrophoretic variant.This work was supported by CNR Contract 76-01961-04.     

Haemonchus contortus: enzymes. 3. Glutamate dehydrogenase

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.     

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.     

Heterologous expression of cDNAs encoding Chlorella sorokiniana NADP-specific glutamate dehydrogenase wild-type and mutant subunits in Escherichia coli cells and comparison of kinetic and thermal stability properties of their homohexamers

Full-length cDNAs encoding the - and -subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant - and -subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The - and -homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic -homohexamer, but it did not display the allosteric response to NADPH shown by the -homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 °C. The temperature optimum for catalytic activity for the - and -homohexamers was 60 °C, and 65 °C for the 40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Localisation and characterisation of homoserine dehydrogenase isolated from barley and pea leaves

Two forms of homoserine dehydrogenase exist in the leaves of both barley and pea; one has a large molecular weight and is inhibited by threonine, the other is of smaller molecular weight and insensitive to threonine but inhibited by cysteine. The subcellular localisation of these enzymes has been examined. Both plants have 60–65% of the total homoserine dehydrogenase activity present in the chloroplast and this activity is inhibited by threonine. The low molecular weight, threonine-insensitive form is present in the cytoplasm. Total homoserine dehydrogenase activity from barley leaves showed progressive desensitisation towards threonine with age in a similar manner to that previously described for maize. It was shown that the effect was due to desensitisation of the chloroplast enzyme, and not to an increase in the insensitive cytoplasm enzyme. No corresponding desensitisation to threonine was detected in pea leaves. The different forms of homoserine dehydrogenase could be separated from pea leaves by chromatography on Blue Sepharose; the threonine-sensitive enzyme passed straight through and the threonine insensitive form was bound. A similar separation of the barley leaf isoenzymes was obtained using Matrex Gel Red A affinity columns; in this case however, the threonine-sensitive isoenzyme was bound. In both plants, the threonine insensitive isoenzyme was subject to greater inhibition by cysteine than was the threonine-sensitive isoenzyme.Abbreviation HSDH homoserine dehydrogenase     

The glutamate dehydrogenase structural gene of Escherichin coli

Summary The glutamate dehydrogenase structural gene, gdhA, was mapped at 38.6 min on the genetic map and at 1860 kb on the physical map. A detailed map of this region is presented.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotrophArthrobacter P1

WhenArthrobacter P₁ is grown on choline, betaine, dimethylglycine or sarcosine, an NAD⁺-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa±10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa±3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the K_m values were 0.10 and 0.80 mM for formaldehyde and NAD⁺, respectively. The enzyme was heat-stable at 50° C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.Abbreviation RuMP Ribulose monophosphate