Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Cloning, mapping, and sequencing of the gene encoding Escherichia coli quinoprotein glucose dehydrogenase.

Escherichia coli contains pyrroloquinoline quinone-dependent glucose dehydrogenase. We cloned and sequenced the gene (gcd) encoding this enzyme and showed that the derived amino acid sequence is highly homologous to that of the gdhA gene product of Acinetobacter calcoaceticus. Stretches of homology also exist between the amino acid sequence of E. coli glucose dehydrogenase and other pyrroloquinoline quinone-dependent dehydrogenases from several bacterial species. The position of gcd on the chromosomal map of E. coli was determined to be at 3.1 min.     

Glutamate dehydrogenases of unicellular green algae: effects of nitrate and ammonium in vivo]

The constitution and control by the inorganic nitrogen source of glutamate dehydrogenases of some unicellular green algae have been studied. The Ankistrodesmus braunii and Scenedesmus obliquus cells contain two different glutamate dehydrogenases, one of which is NADP-specific, the other is active with both NAD and NADP. Their synthesis does not depend on the nitrogen source. The activity of NADP-specific glutamate dehydrogenase increases sharply during nitrogen starvation. In Chlorella pyrenoidosa 82 and Ch. ellipsoidea only one constitutive double specific glutamate dehydrogenase is observed. Its activity does not change depending on the nitrogen nutrition conditions. In the cells of the thermophylic Chlorella strain Chlorella sp. K. ammomium induces a de novo synthesis of NADP-specific glutamate dehydrogenase in addition to the constitutive double specific glutamate dehydrogenase. Thus, the algae tested contain constitutive double specific glutamate dehydrogenase. The NADP-specific enzyme is absent in two Chlorella strains, is constitutive in A. braunii and S. obliquus, and is ammonium-inducible in three thermophylic Chlorella strains.     

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.     

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.     

The gdhA gene is located at 38.6 minutes on the Escherichia coli map.

We report here that the gdhA gene of Escherichia coli, which encodes the NADP-specific glutamate dehydrogenase, is located at 38.6 min on the map. We have confirmed this location by showing linkage with three Tn10 insertions that are linked to the aroD, pheS, and ansA loci, by complementation by a restriction-mapped lambda clone, and by showing correspondence between the restriction maps of the chromosome and the cloned and sequenced gdhA gene.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity

The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. Together with the N-terminal sequence, these make up about 75% of the total sequence. The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence. This demonstrated sequence similarity with the E. coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C. symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked. The evolutionary implications are discussed. In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly. The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.     

Complete nucleotide sequence of the Escherichia coli gdhA gene

The DNA sequence of the gdhA gene of Escherichia coli K12, which encodes the 447 amino acid polypeptide subunit of NADP-specific glutamate dehydrogenase, is presented. The deduced protein sequence is strongly homologous to the corresponding enzyme of the eukaryotic fungus Neurospora crassa. The upstream DNA sequence includes several overlapping promoter consensus sequences. The downstream DNA sequence contains inverted repeats, predicted as forming long stable stem-loop structures in RNA, homologous to those found in several enterobacterial intergenic regions.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.

A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.     

Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)(+)-dependent dehydrogenases

The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined. In addition, the same gene from a moderate thermophile, B. stearothermophilus, was analyzed in a similar manner. Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments. Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme. The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%). The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes. Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain. Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.     

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7.

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.     

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily

We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.     

Cloning,sequence analysis,and heterologous expression of the gene encoding a (<Emphasis Type="Italic">S</Emphasis>)-specific alcohol dehydrogenase from<Emphasis Type="Italic"> Rhodococcus erythropolis</Emphasis> DSM 43297

The gene encoding an (S)-specific NAD-dependent alcohol dehydrogenase (RE-ADH) was isolated from the genomic DNA of Rhodococcus erythropolis DSM 43297. The nucleotide sequence of 1,047 bp, coding for 348 amino acids, was cloned in Escherichia coli cells and successfully expressed. The subunit molecular mass as deduced from the amino acid sequence was determined to be 36.026 kDa. The recombinant enzyme exhibited high thermostability, which facilitated its purification by heat treatment, followed by two column-chromatography steps. RE-ADH shows high similarity to several zinc-containing medium-chain alcohol dehydrogenases. All zinc ligands seem to be conserved except one of the catalytic zinc ligands, where Cys is probably substituted by Asp. A similarity of 84% with a phenylacetaldehyde reductase from Corynebacterium sp. ST-10 was determined. Biochemical properties such as thermostability and substrate specificity of the two enzymes were compared.     

Re-assessment of ammonium-ion affinities of NADP-specific glutamate dehydrogenases. Activation of the Neurospora crassa enzyme by ammonium and rubidium ions.

The NADP-specific glutamate dehydrogenase of Neurospora crassa shows complex interactions with NH4+ ions, characterized by biphasic downwardly convex double-reciprocal plots. These kinetics are explained by the action of NH4+ both as a substrate and, acting at a separate cation-binding site, as an activator. Rb+ ions, and to a smaller extent other univalent cations, also activate by acting as analogues of NH4+. Previous failure to recognize this effect, which probably also occurs in homologous enzymes from some other species, has led to significant overestimates in published reports of the Km for NH4+ of some NADP-specific glutamate dehydrogenases.     

Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases

The genes encoding proteins responsible for activity of the E1 component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined. Open reading frames encoding E1 alpha (bkdA1, 1233 bp) and E1 beta (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits. The Mr of E1 alpha was 45,158 and of E1 beta was 37,007, both calculated without N-terminal methionine. The deduced amino acid sequences of E1 alpha and E1 beta had no similarity to the published sequences of the E1 subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the E1 alpha subunits of Pseudomonas and rat liver branched-chain-oxoacid dehydrogenases. In particular, the region of the E1 alpha subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1 alpha subunit. There was also considerable similarity between the E1 beta subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.     

Glutamate dehydrogenase of Halobacterium salinarum: evidence that the gene sequence currently assigned to the NADP+-dependent enzyme is in fact that of the NAD+-dependent glutamate dehydrogenase

A GDH gene from Halobacterium salinarum has been cloned and sequenced and the publication assigns the sequence to the NADP+-glutamate dehydrogenase of this organism. We have expressed this gene in Escherichia coli and find that it encodes an NAD+-dependent glutamate dehydrogenase without activity towards NADP+. Further, peptide sequence from the two corresponding proteins supports the view that the deposited sequence is indeed that of the NAD+-dependent glutamate dehydrogenase. Sequence from the NAD+-dependent protein matches the published gene sequence, whereas sequence from the NADP+ glutamate dehydrogenase does not.     

A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although alpha-ketoisoleucine was the most favourable of all alpha-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.