Alanine Dehydrogenase Research Articles

Mechanism of alanine excretion in recombinant strains of Zymomonas mobilis

A thiamine-auxotrophic strain of Zymomonas mobilis (CP4thi/pZY73), in which the alaD gene of Bacillus sphaericus coding for the alanine dehydrogenase was expressed, synthesizes and excretes alanine at high rates after thiamine starvation and in the presence of high external ammonium concentrations. The mechanism of alanine excretion was studied in this recombinant Zymomonas mobilis strain. Under production conditions the internal alanine concentration reached values of up to 280 mM and excretion rates of up to 140 nmol min −1 mg dry mass −1 were obtained. The membrane integrity and the energetic properties of the cells remained intact and were comparable to growing wild-type cells. Unspecific leakage of solutes was not observed. We did not find any indication of a carrier-mediated excretion of alanine, since typical properties of this type of mechanism, i.e., saturation at increasing internal substrate concentration, substrate specificity and functional inhibition were absent. Furthermore, a counterflow maximum, which would indicate the involvement of a carrier protein, was not observed either. Consequently, alanine excretion in recombinant Z. mobilis cells is interpreted as mediated by simple diffusion through the intact cytoplasmic membrane at high rates (diffusion constant 10 −8 l s −1 mg dry mass −1 or 0.28 min −1). For comparison, the diffusion constant for alanine efflux was also measured in Corynebacterium glutamicum cells and the values obtained were significantly lower than those determined in Z. mobilis. The consequences of this finding are discussed.

Chemo-enzymatic syntheses of isotopically labelled L-amino acids

L-Alanine labelled with carbon-13 and nitrogen-15 has been prepared in good yield and high optical purity from achiral precursors using alanine dehydrogenase This versatile approach may be simply adapted for the preparation of a range of isotopically labelled amino acids and efficient syntheses of [ 15N]-labelled serine, valine, leucine and phenylalanine are described.

A role for alanine in the ammonium regulation of cephalosporin biosynthesis in Streptomyces clavuligerus.

It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We found L-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two Km values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH4+ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.

The Biochemistry and Enzymology of Amino Acid Dehydrogenases

This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.

Spectrophotometric Assay of Aminopeptidase with Thermostable Alanine Dehydrogenase fromBacillus stearothermophilus

A spectrophotometric assay for aminopeptidase in biological fluids has been developed using a thermostable alanine dehydrogenase (AlaDH, EC 1.4.1.1) from Bacillus stearothermophilus. l-Alanine produced by the aminopeptidase with l-alanine amide or l-leucyl-l-alanine as the substrate, is oxidatively deaminated to pyruvate in the presence of NAD+ by the action of AlaDH. Aminopeptidase activity was continuously monitored by measuring the absorbance at 340 nm corresponding to NADH production. The measured aminopeptidase activity was found to be linear up to 700–800 units/liter at 37°C. The reagents were stable in solution for at least 4 weeks at 10°C. This method was applicable to the assay of serum aminopeptidase. The assays had a high degree of precision even at low aminopeptidase activity and correlated well with the conventional assay methods.

Large-scale production of thermostable alanine dehydrogenase from recombinant cells

We have cloned the thermostable alanine dehydrogenase gene from a thermophile, Bacillus stearothermophilus into Escherichia coli C600 with a vector plasmid pKK 223-3. The recombinant cells produced a large amount of the enzyme, which corresponded to about 10% of the total amount of cellular proteins. In order to prevent the cells from leaking to the environment, cell culture and disruption were consecutively carried out in the same fermentor equipped with a HEPA filter, which served as a biological barrier. The cells grown in the culture broth were treated with Triton X-100, lysozyme, and EDTA, whose final concentrations were 0.1%, 100 mg/ l, and 10 mM, respectively, for 30 min, followed by heating at 60°C for 30 min. Almost all of the enzyme (about 98%) was found in the broth, whereas no living recombinant cells were found in the broth, by these treatments. Based on these we established a large-scale production system of thermostable enzyme from recombinant cells.

Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis.

The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn917 mutation was also found to be an allele of ald. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.

Ammonium assimilation in Nocardia asteroides.

Specific enzymes of ammonium assimilation were measured in cell-free extracts of Nocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation in N. asteroides.

Alanine Dehydrogenase from Soybean Nodule Bacteroids: Purification and Properties

Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH + 4, pyruvate, L-alanine, and NAD were 86 μM, 8.9 mM, 0.49 mM, 1 mM and 200 μM, respectively. High concentrations of pyruvate, L-alanine, or NH + 4 caused inhibition of activity with K i ′s of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible. s. Inc.

Enzymatic synthesis of l-?-chloroalanine using amino acid dehydrogenase

l-β-Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l-β-chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l-β-chloroalanine at pH 7.5. The K m and V max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l-β-Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).

Alanine dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies

The kinetic mechanism of alanine dehydrogenase from soybean nodule bacteroids was studied by initial velocity experiments with or without product inhibitors, dead-end inhibitors, or alternate substrates. Without inhibitors, double-reciprocal plots of initial velocity experiments showed intersecting lines, indicating a sequential mechanism. These initial velocity experiments also revealed rapid-equilibrium ordered binding of NH4+ prior to pyruvate. When NAD was varied at changing-fixed concentrations of L-alanine, a nonlinear, concave down double-reciprocal plot was obtained. Substrate inhibition by pyruvate or L-alanine with cosubstrates varied was uncompetitive giving further support to an ordered mechanism. Product inhibition studies showed that both NAD and NADH and pyruvate and L-alanine were competitive. This suggested a Theorell-Chance mechanism. When product inhibition by L-alanine was studied with NH4+ varied in a series of experiments at increasing concentrations of pyruvate, the inhibition was eliminated, as expected for a Theorell-Chance mechanism. Furthermore, when NADH, NH4+, and pyruvate were varied simultaneously, maintaining their concentrations at a constant ratio to each other, an infinite Vmax was obtained. pH studies of the kinetic parameters indicated that NH4+, rather than NH3, was the true substrate that binds to a residue on the enzyme with a pK of 8.1. In conclusion, the kinetic mechanism at pH 8.5 was determined to be a Ter-Bi Theorell-Chance. In the amination direction, the substrates add in the order: NADH, NH4+, pyruvate, with NH4+ binding in rapid-equilibrium. In the reverse direction, NAD adds first, followed by L-alanine.

Zymoblot, a new microtechnique used to detect enzyme activity in spiroplasmas and bacteria

A new microtechnique that detects enzyme activity in prokaryotes is described. The technique, designated zymoblot, is based on the immobilization of negatively charged enzymes from an alkaline extract spotted onto a nitrocellulose membrane. The presence of specific enzyme activity in the extract is selectively assayed with a reaction mixture containing the corresponding substrate. The enzyme–substrate reaction produces an insoluble colored product that accumulates at the site. The zymoblot technique offers the advantages of simplicity, sensitivity, reproducibility, speed, and the use of microquantities of reactants. The protein in the spot can be visualized by a technique termed "proteinblot," in which the protein is stained with Coomassie blue. Esterase and tyrosinase were detected by the zymoblot method in six spiroplasmas including four strains of Spiroplasma citri, one of Spiroplasma kunkelii, and one of Spiroplasma melliferum, and two bacteria, Pseudomonas syringae pv. syringae B301D and Escherichia coli K-12. Acid phosphatase, alkaline phosphatase, alanine dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase were not detected in any of the spiroplasmas, but were each detected in one or both of the walled bacteria.Key words: spiroplasma, enzyme, protein, zymoblot.

Similarities between Alanine Dehydrogenase and the N-Terminal Part of Pyridine Nucleotide Transhydrogenase and Their Possible Implication in the Virulence Mechanism of Mycobacterium tuberculosis

Recent developments in simultaneous multiple alignment methods of protein sequences allow prediction of structural similarity in related proteins. Alanine dehydrogenase and the N-terminal sequence of pyridine nucleotide transhydrogenase were compared for their sequences. High similarities of sequences were observed especially in their NAD(H)-binding sites. These similarities suggest that antibodies which recognized the alanine dehydrogenase of Mycobacterium tuberculosis can also be directed against the membrane bound pyridine nucleotide transhydrogenase. If this is the case, the virulent property of this pathogen could be linked to its higher synthesis of NADPH necessary for its anabolism.

Stereoselective microbial/enzymatic oxidation of (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding chiral lactol and lactone

A key chiral intermediate, lactol 2[3aS(3aα, 4α, 7α, 7aα)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one, and the corresponding chiral lactone 3 were made in high optical purity by stereoselective enzymatic and microbial oxidation of the parent diol 1. Horse liver alcohol dehydrogenase (HLADH) in the presence of nicotinamide adenine dinucleotide (NAD +) and riboflavin oxidized diol 1 (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding lactol 2 and lactone 3. Regeneration of NAD + required for the oxidation of diol 1 was carried out by the NADH-dependent alanine dehydrogenase and leucine dehydrogenase in the presence of alanine and leucine, respectively. Since expensive enzyme (HLADH) and cofactors (NAD +) were required for the oxidation reaction, various microorganisms were screened for the ability to catalyze the stereoselective oxidation of diol 1. Two organisms, Nocardia globerula ATCC 21505 and Rhodococcus sp. ATCC 15592, catalyzed the efficient oxidation of diol 1 to the corresponding chiral lactol 2 and lactone 3. The reaction yield of 70% and optical purity of 96% was obtained for lactone 3 prepared from the oxidation of diol 1 by cell suspensions (10% w/v, wet cells) of N. globerula ATCC 21505. Substrate was used at 5 gl −1 concentration. A 10% (w/v, wet cells) cell suspension of Rhodococcus sp. ATCC 15592, after 120 h reaction period, produced lactol 2 in 12% yield and lactone 3 in 34% yield. An overall 46% reaction yield was obtained based on diol 1 oxidation. Substrate was used at 5 gl −1 concentration. Optical purity of 96.7% and 98.4% was obtained for lactol 2 and lactone 3, respectively. Lactone 3 purified by slilica gel chromatography gave a specific rotation of +117° and enantiomeric excess of >99%.

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH4 + concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH4 + concentrations. In some characteristics of its NH4 +-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.

Nitrogen fixation and nitrogen metabolism in heliobacteria

Three species of anoxygenic phototrophic heliobacteria, Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis, were studied for comparative nitrogen-fixing abilities and regulation of nitrogenase. Significant nitrogenase activity (acetylene reduction) was detected in all species grown photoheterotrophically on N2, although cells of H. mobilis consistently had higher nitrogenase activity than did cells of either H. chlorum or H. gestii. Nitrogen-fixing cultures of all three species of heliobacteria were subject to “switch-off” of nitrogenase activity by ammonia; glutamine also served to “switch-off” nitrogenase activity but only in cells of H. mobilis and H. gestii. Placing photosynthetically grown heliobacterial cultures in darkness also served to “switch-off” nitrogenase activity. Dark-mediated “switch-off” was complete in lactate-grown heliobacteria but in pyruvate-grown cells substantial rates of nitrogenase activity continued in darkness. In all heliobacteria examined ammonia was assimilated primarily through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway although significant levels of alanine dehydrogenase were present in extracts of cells of H. gestii, but not in the other species. The results suggest that heliobacteria, like phototrophic purple bacteria, are active N2-fixing bacteria and that despite their gram-positive phylogenetic roots, heliobacteria retain the capacity to control nitrogenase activity by a “switch-off” type of mechanism. Because of their ability to fix N2 both photosynthetically and in darkness, it is possible that heliobacteria are significant contributors of fixed nitrogen in their paddy soil habitat.

The relation between the soluble factor associated with H +-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H +-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Th 2) and a membrane-bound component are together required for activity. Th s was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH 4) 2SO 4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an M r of approx. 74 000 under non-denaturing conditions but analysis of the pure proteins by SDS PAGE revealed a single polypeptide, M r 43 000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatographores and decreased the capacity of Th s to restore activity to depleted membranes. They reacted with a polypeptide of M r 43 000 in crude cell extract, chromatophore membranes and chromatophore washing but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43 000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the α subunit of the E. coli protein. The break between the α and β polypeptides of E. coli transhydrogenase is such that both components are membrane associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Th s has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Th s and the reported sequence of alanine dehydrogenase from Bacillus but Th s did not have any alanine dehydrogenase activity.

Enzymatic production of l‐alanine from malic acid with malic enzyme and alanine dehydrogenase with coenzyme regeneration

AbstractAn enzyme reaction system with coenzyme regeneration was investigated for L‐alanine production. Alanine dehyrogenase L‐alanine: NAD+ oxidoreductase (EC 1.4.1.1)] from Corynebacterium flaccumfaciens AHU‐1622 was used as the catalyst for reductive amination of pyruvate to L‐alanine. NAD‐ and NADP‐linked malic enzyme [L‐malate: NAD(P)+ oxidoreductase (EC 1.1.1.39)] from Pseudomonas diminuta IFO‐13182 was used for the regeneration of NADH. Optimum conditions for L‐alanine production were determined, including L‐malic acid concentration, MgCl2 concentration and pH. Under suitable conditions, the conversion of L‐malic acid to L‐alanine reached 95% after 72 h of incubation at 30°C, yielding 106 mol/m3 of L‐alanine. The L‐alanine produced was purified in crystal form; its purity was 99.4%, based on HPLC analysis.

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.