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At least 19 recordsLinked to original sources
PubMed2005

Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease.

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.

Alanine↗
PubMed1976

A transition state analogue for two pyruvate metabolizing enzymes, lactate dehydrogenase and alanine dehydrogenase.

The synthesis of 5-(2-oxalylethyl)-NADH, a reduced nicotinamide adenine dinucleotide (NADH) derivate with pyruvate covalently attached to the 5 position of the dihydronicotinamide ring over an additional methylene group has been described previously (Trommer, W.E., Blume, H., and Kapmeyer, H. (1976) Justus Liebigs Ann. Chem., 848). In the presence of lactate dehydrogenase, the dihydropyridine ring of this coenzyme-substrate analogue is oxidized and the carbonyl function of the side chain is reduced to the corresponding L-hydroxy derivative with a maximum velocity of 1/3000 of the natural reaction. This reaction is intramolecular as shown by competition experiments with pyruvate. 5-(2-oxalylethyl)-NADH (pyr-NADH) appears to be a true transition state analogue, proving its postulated structure. Pyr-NADH is high specific for this enzyme as demonstrated by the facts that (1) D-lactate dehydrogenase does not catalyze the intramolecular redox reaction, although the substrate moiety of pyr-NADH is reduced in the presence of NADH; (2) when tested with malate dehydrogenase, alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase,glycerate dehydrogenase, and glycerol dehydrogenase pyr-NADH is not even oxidized in the presence of the corresponding substrates. However, a great similarity between the transition states of the reduction of pyruvate catalyzed by lactate dehydrogenase and alanine dehydrogenase could be shown. Alanine dehydrogenase catalyzes the intramolecular redox reaction as well. In the presence of ammonium ions, pyr-NADH is transformed to 5-(3-carboxyl-3-aminopropyl)-NAD+.

Alanine↗
PubMed1987

Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.

Alanine Dehydrogenase↗
PubMed1989

Partial purification and properties of Cunninghamella elegans L-alanine dehydrogenase.

L-Alanine dehydrogenase was partially purified from the mycelial extracts of Cunninghamella elegans. The purified enzyme was fractionated by TEAE-cellulose column chromatography into two fractions (subunits) designated as fractions I and II. The activity of both fractions in the aminating reaction is 8 times higher than the activity of the deaminating reaction. pH-Optimal for reductive amination of pyruvate by both fractions were 8 and 10 for oxidative deamination of L-alanine. The Km values of fractions I and II for L-alanine, NAD+, pyruvate, NH4+ and NADH were estimated. Both fractions of L-alanine dehydrogenase were absolutely specific for L-alanine in the oxidative deamination reaction. Maximal activity of both fractions occurred at 40 degrees C. Inhibition by iodoacetate and activation by SH-compounds suggest that sulfhydryl groups may participate in enzyme activity. The 2 fractions were activated with Co2+, Fe2+, Ca2+, Mg2+ and Mn2+ ions, while Zn2+ inhibited both fractions. Stability of the enzyme under different conditions was investigated.

Alanine↗
PubMed1990

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.

Alanine Dehydrogenase↗
PubMed2002

Screening and simple purification of alanine dehydrogenase in Bacillus strains.

Alanine dehydrogenase (EC 1.4.1.1), in the presence of NAD+, catalyzes the reversible deamination of L-alanine. Screening of alanine dehydrogenase in bacillus strains was carried out to develop its utilization as an industrial and analytical catalyst. Eight bacillus strains were used, including Bacillus megaterium LA 199 which abundantly produces enzymes. Alanine dehydrogenase was purified simply from Bacillus megaterium LA 199 by heat treatment at pH 5.4, followed by DEAE-Sepharose CL-6B and Sepharose CL-2B chromotography. The enzyme consisted of six subunits with an identical molecular mass of 42.5 kDa. The Km were 1.17 x 10(-2) mM for NADH and 5.12 x 10(-2) mM for pyruvate.

Alanine Dehydrogenase↗
PubMed1981

Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase.

L-Alanine dehydrogenase from Bacillus subtilis has a predominately ordered kinetic mechanism in which NAD adds before L-alanine, and ammonia, pyruvate, and NADH are released in that order. When pyruvate is varied at pH 9.35, levels of ammonia above 50 mM cause uncompetitive substrate inhibition and cause the slope replot to go through the origin. This pattern suggest that iminopyruvate (2% of pyruvate at this pH with 150 mM ammonia) can combine with E-NADH much more tightly than pyruvate does but reacts much more slowly because uptake of the required proton from solution is hindered. Isomerization of the initially formed E-NAD complex to a form which can productively bind L-alanine is the slowest step in the forward direction at pH 7.9, and substrate inhibition by L-alanine largely results from combination of the zwitterion in a nonproductive fashion with this initial E-NAD complex, with the result that the isomerization is prevented. All bimolecular rate constants approach diffusion-limited values at optimal states of protonation of enzyme and substrates except that for ammonia, suggesting that ammonia does not form a complex with E-NADH-pyruvate but reacts directly with it to give a bound carbinolamine.

Alanine Dehydrogenase↗
PubMed1976

Regulatory control and function of alanine dehydrogenase from a thermophilic bacillus.

L-alanine dehydrogenase, (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) synthesis in a thermophilic bacillus was found to be subjected to regulatory control. Addition of L- and D-alanine and L-serine to cultures growing in the presence of either succinate or pyruvate, induced an accelerated synthesis of the alanine dehydrogenase enzyme. Synthesis of the enzyme was dependent on the presence of inducer during growth and was arrested by addition of glucose. Catabolite repression by glucose was abolished by limiting the ammonium concentration during growth. The apparent Km values of the substrates involved in alanine dehydrogenase activity are as follows (M): NH4+, 4-10(-2); pyruvate, 5-10(-4); NADH, 6-10(-5); L-alanine, 3.1-10(-3) and NAD, 2-10(-4). Alanine dehydrogenase activity was measurable at temperatures below the minimal growth temperature (at 25 degrees C) and the highest activity was found at 65 degrees C; heat denaturation occurred at 80 degrees C.

Alanine↗
PubMed1983

Catalytic properties of Sepharose-bound L-alanine dehydrogenase from Bacillus cereus.

(1) L-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, L-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH4+, pyruvate and L-alanine, was not different to that of the soluble form. The Km of the Sepharose-bound L-alanine dehydrogenase for pyridine coenzymes was 6-8-times higher than in the soluble case. (3) The stability of L-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37 degrees C for 24 h of the immobilized L-alanine dehydrogenase with 3 M NH4Cl/NH4OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the 'solubilized' L-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble L-alanine dehydrogenase.

Alanine Dehydrogenase↗
PubMed1998

[Purification and properties of alanine dehydrogenase from Streptomyces lincolnensis].

Alanine Dehydrogenase (L-Alanine: NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was purified from Streptomyces lincolnensis through four steps: (NH4)2SO4 precipitation, DEAE-cellulose 52, Affi-Gel Blue and Sepharose 6B. Molecular weight of the enzyme was determined as 170,000 by gel filtration and concentration gradient PAGE. SDS-PAGE showed only one band of 42,500, demonstrating that ADH from Streptomyces lincolnensis was consisted of four identical subunits. The optimal pH for amination was 9.0, for deamination 9.5. The optimal temperature for both amination and deamination was 50 degrees C. The Km valuse for pyruvate, NH4+, NADH, L-Ala and NAD+ were 2.08 x 10(-4) mol/L, 2.00 x 10(-2) mol/L, 2.38 x 10(-5) mol/L, 1.43 x 10(-2) mol/L and 6.67 x 10(-5) mol/L, respectively.

Alanine Dehydrogenase↗
PubMed1993

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.

Alanine Dehydrogenase↗
PubMed2000

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia.

Alanine dehydrogenase [L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent Km values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.

Alanine Dehydrogenase↗
PubMed1991

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.

Alanine↗
PubMed1975

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.

Alanine↗