Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.     

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 from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

Alanine dehydrogenase and its applications – A review

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD⁺/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.     

Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1.

In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

A novel archaeal alanine dehydrogenase homologous to ornithine cyclodeaminase and mu-crystallin

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.     

A new NAD+-dependent opine dehydrogenase from Arthrobacter sp. strain 1C.

A new NAD+-dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique. The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with molecular weights of about 36,000. The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme was active toward unusual opines, such as N-[1-R-(carboxyl)ethyl]-S-methionine and N-[1-R-(carboxyl)ethyl]-S-phenylalanine. In the reductive secondary amine-forming reaction with NADH as a cofactor, the enzyme utilized L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-leucine, L-alanine, and L-threonine as amino donors and alpha-keto acids such as pyruvate, oxaloacetate, glyoxylate, and alpha-ketobutyrate as amino acceptors. The product enzymatically synthesized from L-phenylalanine and pyruvate in the presence of NADH was identified as N-[1-R-(carboxyl)ethyl]-S-phenylalanine.     

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.     

Room-temperature synthesis of L-alanine using the alanine dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Alanine dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-alanine at room temperature was accompanied by no detectable loss of alanine dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.     

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.     

Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobe Bilophila wadsworthia.

Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mM, Vmax = 1.20 nmol.s-1, and for pyruvate: Km = 0.82 mM, Vmax = 0.17 nmol.s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.     

Continuous production of 3-fluoro-L-alanine with alanine dehydrogenase

Alanine dehydrogenase catalyzed the conversion of 3-fluoropyruvate into 3-fluoro-L-alanine in the presence of NADH and ammonia. The optimum pH of the reaction was 7.8. The K(m) values of the enzyme for 3-fluoropyruvate, polyethylene glycol-bound NADH, and ammonia were 2.94, 0.56, and 105mM, respectively. 3-Fluoro-L-alanine was selectively and continuously produced from 3-fluoropyruvate and ammonium formate in an enzyme membrane reactor by the multienzyme reaction system of alanine dehydrogenase and formate dehydrogenase with a simultaneous coenzyme regeneration. The average conversion and the space-time yield were 73% and 75 g/L day, respectively, with operation of the reactor for 4 days. Alanine dehydrogenase and formate dehydrogenase consumed were 11, 370 and 22, 950 units/kg 3-fluoro-L-alanine, respectively. The cycle number was 3150 mol/mol NAD.     

A spectrophotometric rate assay of aminoacylase

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.     

Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.