Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 X 10(-2) and 9 X 10(-5) M, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.     

A mutant of Aspergillus nidulans defective in NAD-linked glutamate dehydrogenase.

A mutation leading to partial loss of NAD-linked ("catabolic') glutamate dehydrogenase does not affect the regulation of ammonium-repressible activities in Aspergillus nidulans. This mutation has been used to show that NAD-linked glutamate dehydrogenase does not normally participate in ammonium assimilation. A mutation leading to loss of NADP-linked ("anabolic') glutamate dehydrogenase has been used to show that NADP-linked glutamate dehydrogenase is not normally involved in glutamate catabolism. Strains defective in either enzyme are useful for determining which amino acids are metabolised via transamination to yield glutamate rather than via deamination to yield ammonium.     

Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.     

Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase.

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.     

Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli

To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.     

Purification of pantoate and dimethylmalate dehydrogenase from Pseudomonas fluorescens UK-1.

Pantoate dehydrogenase and dimethylmalate dehydrogenase were purified 69- and 112-fold, respectively, from Pseudomonas fluorescens UK-1 by ammonimu sulphate precipitation. Ultrogel AcA 34 gel filtration, hydroxyapatite column chromatography, heat treatment and Ultrogel AcA 44 gel filtration. The enzymes were evaluated for homogeneity (pantoate dehydrogenase was estimated to be about 95% pure) by disc and sodium dodecyl sulphate gel electrophoresis and by immunodiffusion. Pantoate and dimethylmalate dehydrogenases have molecular weights of 83 000 and 138 000, respectively, and are dissociable into four identical subunits with molecular weights of 24 000 and 34 000.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

Conversion of L-Hydroxyproline to glutamate by extracts of strains of Pseudomonas convexa and Pseudomonas fluorescens

Summary Three strains of Pseudomonas convexa and three strains of Pseudomonas fluorescens were found able to utilize L-hydroxyproline as sole source of carbon and nitrogen. Sonic extracts of these organisms converted L-hydroxyproline to glutamic acid.     

Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species

A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.     

Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of 相似文献   

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

Possible physiological roles of aspartase, NAD- and NADP-requiring glutamate dehydrogenases of Pseudomonas fluorescens.

The levels of aspartase, NADP- and NAD-requiring glutamate dehydrogenases (GDHs) in Pseudomonas fluorescens grown under various nutritional conditions were determined. NADP-GDH showed the highest value on glucose-ammonium sulfate medium and markedly lower values on amino-acid and casamino-acids media, while the reverse was found for the NAD-GDH, as in the case of other microorganisms with two GDHs. Aspartase did not show a marked variation between the media examined. Glucose nutritionally induced NADP-GDH but suppressed NAD-GDH; and it had no effect on aspartase, which was slightly induced by casamino acids. Transfer of the cells grown on glucose-ammonium sulfate medium to casamino-acids medium clearly increased the levels of NAD-GDH and aspartase, while addition of chloramphenicol to the media abolished the increases, suggesting that the increases were due to de novo synthesis of the enzyme proteins. These results indicate that the aspartase of this microorganism has a different function from those in others, including Escherichia coli.