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.     

Thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus: gene and protein sequence determination and relationship to other alcohol dehydrogenases.

The NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) from the thermoacidophilic archaebacterium Sulfolobus solfataricus, DSM1617 strain (SSADH), has been purified and characterized. Its gene has been isolated by screening two S. Solfataricus genomic libraries using oligonucleotide probes. The encoding sequence consists of 1041 base pairs, and it shows a high preference for codons ending in T or A. The primary structure, determined by peptide and gene analysis, consists of 347 amino acid residues, yielding a molecular weight of 37,588. A level of identity of 24-25% was found with the amino acid sequences of horse liver, yeast, and Thermoanaerobium brockii alcohol dehydrogenases. The coenzyme-binding and catalytic and structural zinc-binding residues typical of eukaryotic alcohol dehydrogenases were found in SSADH with the difference that one out of the four structural zinc-binding Cys residues is substituted by Glu. The protein contains four zinc atoms per dimer, two of which are removed by chelating agents with a concomitant loss of structural stability.     

Evolutionary relationship of NAD(+)-dependent D-lactate dehydrogenase: comparison of primary structure of 2-hydroxy acid dehydrogenases.

A comparison of the primary structures of NAD(+)-dependent D-lactate dehydrogenase with L-lactate dehydrogenase and L-malate dehydrogenase failed to show any sequence similarity. However, D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, glycerate dehydrogenase from cucumber, D-3-phosphoglycerate dehydrogenase and erythronate 4-phosphate dehydrogenase from Escherichia coli showed 38%, 24%, 24% and 22% amino acid identity, respectively. The profile analysis of the aligned sequences confirmed their relatedness. The hydropathy profiles of the aligned dehydrogenases were almost identical between residues 100-300 indicating largely preserved folding patterns of their polypeptide chains. The data suggest that L- and D-specific 2-hydroxy acid dehydrogenase genes evolved from two different ancestors and thus represent two different sets of enzyme families.     

Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases.

The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit M(r) of around 50,000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with M(r) 115,000. Sequence comparisons have indicated a low level of similarity between the C-terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three-dimensional structure of the hexameric NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C-terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three-dimensional structure and points to a remarkably similar layout of the glutamate-binding site and the active-site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.     

Sequence comparisons betweenAgrobacterium tumefaciens T-DNA-encoded octopine and nopaline dehydrogenases and other nucleotide-requiring enzymes: Structural and evolutionary implications

Summary The nucleotide sequences of the two T-DNA-encoded crown gall imino acid dehydrogenases octopine dehydrogenase and nopaline dehydrogenase were compared with each other and with the sequences of other dehydrogenases. A multistep strategy comprising computer sequence analysis and secondary- and antigenic-structure predictions was used. An alignment of octopine and nopaline dehydrogenase was obtained in which a 20-amino-acid N-terminal arm and six fairly long gaps in the C-terminal moiety were introduced. The aligned sequences have identities of 26% at the amino acid level and 38% at the nucleotide level. They appear to contain two domains. The N-terminal coenzyme-binding domains are similar to those of the well-characterized NAD(P) dehydrogenases. Conserved fragments were found in the C-terminal catalytic domains that likely contain essential residues for catalysis. Comparison of the sequences with those of two other 2-keto acid dehydrogenases, lactate and malate dehydrogenase, suggests that as in those enzymes, histidine, aspartic acid, and arginine residues are located at the octopine and nopaline dehydrogenase active sites. The crown gall enzymes could not be classified with any known family of dehydrogenases. Their evolutionary origin remains unknown. However, predictions concerning their internal organization may provide new insight into protein evolution.     

Cloning and sequence analysis of a cDNA for 3-hydroxyisobutyrate dehydrogenase. Evidence for its evolutionary relationship to other pyridine nucleotide-dependent dehydrogenases

A 1.7-kilobase pair cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase has been isolated by screening a rat liver lambda gt11 library with a 17-base oligonucleotide probe which corresponds to a portion of the N-terminal amino acid sequence of rabbit liver 3-hydroxyisobutyrate dehydrogenase. The cDNA contains an open reading frame of 1038 base pairs which includes an amino acid sequence that matches the N-terminal 35 amino acid sequence of rabbit 3-hydroxyisobutyrate dehydrogenase at 33 residues. The cDNA predicts a 300-amino acid mature protein with an amino acid composition and molecular weight very similar to that of rabbit liver 3-hydroxyisobutyrate dehydrogenase. Northern blot analysis of total RNA from several rat tissues shows an mRNA of approximately 2.0 kilobase pairs in each tissue. Relative mRNA levels were: kidney greater than liver = heart greater than muscle. The amino acid sequence of 3-hydroxyisobutyrate dehydrogenase shows similarity to several other pyridine nucleotide-dependent dehydrogenases. The resemblance to malate and lactate dehydrogenases suggests that the nucleotide-binding domain is located in the N-terminal region of the protein.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

Malate dehydrogenase from the mesophile Chlorobium vibrioforme and from the mild thermophile Chlorobium tepidum: molecular cloning, construction of a hybrid, and expression in Escherichia coli.

The genes (mdh) encoding malate dehydrogenase (MDH) from the mesophile Chlorobium vibrioforme and the moderate thermophile C. tepidum were cloned and sequenced, and the complete amino acid sequences were deduced. When the region upstream of mdh was analyzed, a sequence with high homology to an operon encoding ribosomal proteins from Escherichia coli was found. Each mdh gene consists of a 930-bp open reading frame and encodes 310 amino acid residues, corresponding to a subunit weight of 33,200 Da for the dimeric enzyme. The amino acid sequence identity of the two MDHs is 86%. Homology searches using the primary structures of the two MDHs revealed significant sequence similarity to lactate dehydrogenases. A hybrid mdh was constructed from the 3' part of mdh from C. tepidum and the 5' part of mdh from C. vibrioforme. The thermostabilities of the hybrid enzyme and of MDH from C. vibrioforme and C. tepidum were compared.     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.      

Tauropine dehydrogenase from the marine sponge Halichondria japonica is a homolog of ornithine cyclodeaminase/mu-crystallin

The partial amino acid sequence including the N- and C-terminal portions of tauropine dehydrogenase (EC 1.5.1.23) from the marine sponge Halichondria japonica was determined by enzymatic cleavages followed by peptide sequencing. This information was used to design degenerate primers for amplification of cDNA encoding the tauropine dehydrogenase. The cDNA included 1231 nucleotides with an open reading frame of 1002 nucleotides that encodes a protein of 334 amino acid residues. From the peptide and nucleotide sequencing, the mature tauropine dehydrogenase was estimated to consist of 333 amino acid residues with an acetylated N-terminal serine residue and no intrachain disulfide bonds. The primary structure of the H. japonica enzyme showed apparent similarity with a homolog of ornithine cyclodeaminase from Rhizobium meliloti and other proteins of the ornithine cyclodeaminase/mu-crystallin family, but it showed no significant similarity with the known sequences of octopine dehydrogenases and tauropine dehydrogenases from marine invertebrates. These findings indicate that opine dehydrogenases in marine invertebrates are not all homologous.     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases.

We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength.     

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.     

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.     

Extended superfamily of short alcohol-polyol-sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25% sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcohol-polyol-glucose dehydrogenases.     

The primary structure of Escherichia coli L-threonine dehydrogenase

The complete primary structures of Escherichia coli L-threonine dehydrogenase has been deduced by sequencing the cloned tdh gene. The primary structure so determined agrees with results obtained independently for the amino acid composition, the N-terminal amino acid sequence (20 residues), and a short sequence at the end of an internal peptide of the purified enzyme. The presence of a predicted Asp-Pro bond at residues 148 and 149 was confirmed by treatment of purified threonine dehydrogenase with dilute acid and subsequent analysis of the resulting cleavage products. The primary structure of L-threonine dehydrogenase from E. coli has been examined for possible homology to other NAD+-dependent dehydrogenases; indications are that this enzyme is a member of the zinc-containing long-chain alcohol/polyol dehydrogenase family.     

Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius: cloning, expression, and sequencing of its gene

The gene encoding the thermostable phenylalanine dehydrogenase [EC 1.4.1.-] of a thermophile, Thermoactinomyces intermedius, was cloned and its complete DNA sequence was determined. The phenylalanine dehydrogenase gene (pdh) consists of 1,098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41,000) of the hexameric enzyme. The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T. intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehydrogenases of Bacillus sphaericus and Sporosarcina ureae, respectively. It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B. stearothermophilus. The pdh gene was highly expressed in E. coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein. We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.