2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family

An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 ± 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher V_max/K_m. When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5. The optimum temperature was 52 ºC and at 65 ºC there was a pronounced activity decrease. This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.     

Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea

Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (-)-threo-isohomocitrate [(-)-threo-1-hydroxy-1,2, 4-butanetricarboxylic acid], (-)-threo-iso(homo)(2)citrate [(-)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (-)-threo-iso(homo)(3)citrate [(-)-threo-1-hydroxy-1,2, 6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be alpha-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100 degrees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80 degrees C for 10 min.     

Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137

A halophilic NAD⁺-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD⁺ as a coenzyme and used -2-aminobutyrate, -alanine, and -norvaline as substrates. The best substrate was -2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of -2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for -2-aminobutyrate, 0.16 mM for NAD⁺, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The K_m values for the substrates depended on the concentration of KCl, and the K_m values decreased under high salt conditions.     

Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement

The single amino acid replacement of Tyr52 with Leu drastically increased the activity of Lactobacillus pentosus NAD-dependent D-lactate dehydrogenase toward larger aliphatic or aromatic 2-ketoacid substrates by 3 or 4 orders of magnitude and decreased the activity toward pyruvate by about 30-fold, converting the enzyme into a highly active D-2-hydroxyisocaproate dehydrogenase.     

Metabolism of 2-oxoaldehydes in yeasts. Purification and characterization of lactaldehyde dehydrogenase from Saccharomyces cerevisiae

NAD-dependent lactaldehyde dehydrogenase, catalyzing an oxidation of lactaldehyde to lactate, was purified approximately 70-fold from cell extracts of Saccharomyces cerevisiae with a 28% yield of activity. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The relative molecular mass of the enzyme was estimated to be 40 000 on Sephadex G-150 column chromatography and on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5, 60 degrees C and specifically oxidized L-lactaldehyde to L-lactate in the presence of NAD. The Km values for L-lactaldehyde and NAD were 10 mM and 2.9 mM, respectively. The purest enzyme was extremely unstable and almost completely inactivated during storage at -20 degrees C, pH 7.5. For the reactivation of the enzyme, halide ions such as Cl-, I- and Br- were required.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus

A human fecal isolate, characterized by morphological, physiological and biochemical data as a strain of Peptostreptococcus roductus, was shown to contain NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and a NADP-dependent 7 beta-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were synthesized constitutively. Specific enzymatic activities were significantly reduced when bacteria were grown in the presence of 3-keto bile acids, while other bile acids were ineffective. For the 3 alpha (3 beta)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95,000 (132,000) was estimated. 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50 degrees C for 10 min). The 7 beta-hydroxysteroid dehydrogenase was already present in uninduced cells, but specific activity could be enhanced up to more than 2.5-fold when bacteria were grown in the presence of 7-keto bile acids. Disubstituted bile acids were more effective than trisubstituted ones, ursodeoxycholic acid was ineffective as an inducer. A pH optimum of 10.0 and a molecular weight of about 82,000 were shown for the 7 beta-hydroxysteroid dehydrogenase. The enzyme preparation reduced the 7-keto group of corresponding bile acids. Again the affinities of disubstituted bile acids for the enzyme were higher than those of the trisubstituted bile acids, but no significant differences between conjugated and free bile acids were observed. The 7 beta-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50 degrees C for 10 min), but was detectable at 4 degrees C for at least 48 h.     

D-(+)-lactate dehydrogenase from Lactobacillus murinus

D-(+)-Lactate dehydrogenase from Lactobacillus murinus was purified 670-fold. The Mr was 140,000 as determined by gel filtration. Maximum enzymatic activity was observed at 25 degrees C and pH 6.0 in 200 mM Na2KPO4 buffer. When the temperature was increased from 60 to 65 degrees C, the enzyme was completely inactive in 5 min. The apparent Km for pyruvate and NADH were 4.7 x 10(-4) and 1 x 10(-5) M, respectively. Pyruvate analogs such as oxalate, oxamate, 2-oxobutyrate, and malonate acted as a competitive inhibitors. L-Lactate and L-malate were noncompetitive inhibitors.     

Purification and characterization of glucose dehydrogenase from a heterotrophically grown blue-green alga

NADP- and NAD-dependent glucose dehydrogenase was partially purified from a dark-grown blue-green alga (endophytic Nostoc strain MAC). Polyacrylamide gel electrophoresis established that a single protein possessed dual activity for either NADP or NAD. No other electron acceptor substituted for pyridine nucleotides and no evidence for a flavin prosthetic group was found. Although the Km for NADP was 8.8 mum and for NAD 328 mum, the enzyme was equally active with NAD or NADP at saturating levels of substrates. The enzyme was similar to previously described glucose dehydrogenase in that it had a high Km for glucose (18-20 mm at 35 C) and an alkaline pH optimum of 7.6 to 9.4.     

L-erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase

Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.     

The effect of diabetes, nutritional factors, and sex on rat liver and kidney mevalonate kinase, mevalonate-5-phosphate kinase, and mevalonate-5-pyrophosphate decarboxylase

Isopycnic sucrose gradient separation of rat liver organelles revealed the presence of two distinct branched-chain α-keto acid decarboxylase activities; a mitochondrial activity, which decarboxylates the three branched-chain α-keto acids and requires CoA and NAD⁺ and a cytosolic activity, which decarboxylates α-ketoisocaproate, but not α-ketoisovalerate, or α-keto-β-methylvalerate. The latter enzyme does not require added CoA or NAD⁺. Assay conditions for the cytosolic α-ketoisocaproate decarboxylase activity were optimized and this activity was partially characterized. In rat liver cytosol preparations this activity has a pH optimum of 6.5 and is activated by 1.5 m ammonium sulfate. The decarboxylase activity has an apparent K_m of 0.03 mm for α-ketoisocaproate when optimized assay conditions are employed. Phenylpyruvate is a very potent inhibitor. α-Ketoisovalerate, α-keto-β-methylvalerate, α-ketobutyrate, and α-ketononanoate also inhibit the α-ketoisocaproate decarboxylase activity. The data indicate that the soluble α-ketoisocaproate decarboxylase is an oxidase. Rat liver cytosol preparations consumed oxygen when either α-ketoisocaproate or α-keto-γ-methiolbutyrate were added. None of the other α-keto acids tested stimulated oxygen consumption. 1-¹⁴C-Labeled α-keto-γ-methiolbutyrate is also decarboxylated by cytosol preparations. The α-ketoisocaproate oxidase was purified 20-fold from a 70,000g supernatant fraction of a rat liver homogenate. In these preparations the activity was increased 4-fold by the addition of dithiothreitol, ferrous iron, and ascorbate. The major product of this enzyme activity is β-hydroxyisovalerate. Isovalerate is not a free intermediate in the reaction. The data indicate an alternative pathway for metabolism of α-ketoisocaproate which produces β-hydroxyisovalerate.     

The first thermophilic alpha-oxoamine synthase family enzyme that has activities of 2-amino-3-ketobutyrate CoA ligase and 7-keto-8-aminopelargonic acid synthase: cloning and overexpression of the gene from an extreme thermophile, Thermus thermophilus, and characterization of its gene product

The first thermophilic alpha-oxoamine synthase family enzyme was identified. The gene (ORF TTHA1582), which is annotated to code putative alpha-oxoamine synthase family enzymes, 7-keto-8-aminopelargonic acid (KAPA) synthase (BioF, 8-amino-7-oxononanoate synthase, EC 2.3.1.47) and 2-amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29), in a genomic database, was cloned from an extreme thermophile, Thermus thermophilus, and overexpressed in Escherichia coli. The recombinant TTHA1582 protein was purified and characterized. It exhibited activity of BioF, which catalyzes the condensation of pimeloyl-CoA and L-alanine to produce a biotin intermediate KAPA, CoASH, and CO(2) with pyridoxal 5'-phosphate as a cofactor. The protein is a dimer with a subunit of 43 kDa that shows an amino acid sequence identity of 35% with E. coli BioF. The optimum temperature and pH were about 70 degrees C and about 6.0. The enzyme showed high thermostability at temperatures of up to 70 degrees C for 1 h, and a half-life of 1 h at 80 degrees C. Thus the TTHA1582 protein was found to have the highest optimum temperature and thermostablility of the alpha-oxoamine synthase family enzymes so far reported. Substrate specificity experiments revealed that it was also able to catalyze the KBL reaction, which used acetyl-CoA and glycine as substrates, and that enzyme activity was seen with the following combinations of substrates: acetyl-CoA and glycine, L-alanine, or L-serine; pimeloyl-CoA and L-alanine, glycine, or L-serine; palmitoyl-CoA and L-alanine. This suggests that the recombinant TTHA1582 protein has broad substrate specificity, unlike the reported mesophilic enzymes of the alpha-oxoamine synthase family.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M,and cloning of the gene

Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.     

Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1

An NAD-dependent secondary alcohol dehydrogenase (ADH) produced by Rhodococcus sp. GK1 was purified about fivefold with a yield of 82% by hydrophobic interaction chromatography. This enzyme reduced monoketones, diketones and α-dicarbonyl compounds ; it oxidized secondary alcohols but not primary alcohols. Optimum pH was 7·0 or 8·5 for reduction or oxidation of substrates, respectively, and optimal temperature for activity was 55 °C. The apparent molecular mass of ADH was about 60 kDa by gel filtration chromatography.     

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.     

l-leucine dehydrogenase fromBacillus cereus

Summary An improved method for the production ofl-leucine dehydrogenase is described employing a mutant with a constitutive enzyme and a fed-batch cultivation technique yielding high cell concentrations. Purification ofl-leucine dehydrogenase to homogeneity was carried out starting with 30 kgBacillus cereus cells by heat treatment at 63°C, followed by two liquid-liquid extraction steps and three conventional column chromatographies. Crystals have been obtained from the 95-fold purified enzyme. The molecular weight of the native enzyme was determined by sedimentation equilibrium and gel filtration studies to be 310 000 containing eight identical subunits with a molecular weight of 39 000. The sedimentation coefficient was estimated to 11.65 S. Branched-chain amino acids likel-leucine,l-valine orl-isoleucine are deaminated by the NAD-dependent enzyme. In the reverse reaction a variety of 2-ketoacids, especially 2-ketoisocaproate, 2-ketoisovalerate and 2-keto-3-methyl-valerate, were reductive aminated to the correspondingl-amino acids in the presence of 0.9 M ammonia. The amino acid composition for the subunit ofl-leucine dehydrogenase is presented.     

Analysis of bacterial glucose dehydrogenase homologs from thermoacidophilic archaeon Thermoplasma acidophilum: finding and characterization of aldohexose dehydrogenase

The NADP(+)-preferring glucose dehydrogenase from thermoacidophilic archaeon Thermoplasma acidophilum has been characterized, and its crystal structure has been determined (Structure, 2:385-393, 1994). Its sequence and structure are not homologous to bacterial NAD(P)(+)-dependent glucose dehydrogenases, and its molecular weight is also quite defferent. On the other hand, three functionally unknown genes with homologies to bacterial NAD(P)(+)-dependent glucose dehydrogenases have been sequenced as part of the T. acidophilum genome project (gene names: Ta0191, Ta0747, and Ta0754 respectively). We expressed two genes of three, Ta0191 and Ta0754, in Escherichia coli, and purified the gene products to homogeneity. Dehydrogenase activities were thereby detected from the purified proteins. The Ta0754 gene product exhibited aldohexose dehydrogenase activity, and the Ta0191 gene product exhibited weak 2-deoxyglucose dehydrogenase activity. No aldohexose dehydrogenase gene has been isolated, while the enzyme was reported in 1968. This is the first report of the gene and primary structure. The purified Ta0754 gene product, designated AldT, was characterized. The enzyme AldT effectively catalyzed the oxidation of various aldohexoses, especially D-mannose. Lower activities on D-2-deoxyglucose, D-xylose, D-glucose, and D-fucose were detected although no activities were shown on other aldohexoses or additional sugars. As a cofactor, NAD(+) was much more suitable for the activity than NADP(+). The NAD(+)-preferring dehydrogenase most effectively reacting to D-mannose is for the first time. AldT was most active at pH 10 and above 70 degrees C, and completely stable up to 60 degrees C after incubation for 15 min. Other enzymatic properties were also investigated.     

Purification and characterization of aromatic amine dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on beta-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (alpha2beta2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the alpha-subunit (42.3-kDa subunit) and the beta-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the beta-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and beta-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 degrees C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, beta-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 microM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 degrees C for one month at least in phosphate buffer (pH 7.0).     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.