J. Biochem. 116, 995-1000 (1994) Purification and Characterization of Alanine Dehydrogenase from a Cyanobacterium, Phormidium lapideum Yoshihiro Sawa,1 Masaaki Tani, Ken Murata, Hitoshi Shibata, and Hideo Ochiai Department of Applied Biochemistry, Faculty of Agriculture, Shimane University, Matsue, Shimane 690 Received for publication, May 12, 1994 Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0mM for L-alanine and 0.04mM for NAD+, 0.33mM for pyruvate, 60.6mM for NH4+ (pH 8.7), and 0.02mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5•L-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP. Key words: alanine dehydrogenase, cyanobacterium, Phormidium lapideum. Alanine dehydrogenase [L-alanine: NAD+ oxidoreductase, enzymological and regulatory aspects of AlaDH from deaminating, EC 1.4.1.1] catalyzes a reversible oxidative cyanobacteria, we have purified the enzyme from a non deamination of L-alanine to pyruvate and is found in -N2-fixing thermophilic cyanobacterium, Phormidium lapi Bacillus species and some other bacteria (1, 2). The deum, isolated from Matsue hot springs, Japan. In this enzymological and kinetic properties of the enzyme purified paper, we describe the purification, catalytic and structural from various bacteria have been elucidated (3-9). Recent properties of the enzyme from P. lapideum and discuss its ly, several bacterial AlaDH genes have been cloned and role in the amino acid metabolism in non-N2-fixing cyano their primary structures determined (10-12). bacterial cells. AlaDH has an important role in the carbon and nitrogen metabolism of various microorganisms by providing a link MATERIALS AND METHODS between carbohydrate and amino acid metabolisms (13). In contrast to GluDH, AlaDH occurs only in a limited number Chemicals-NAD+, NADP+, NADH, NADPH, and of bacterial species. Apparently Bacillus AlaDH is involved molecular mass standards for gel filtration chromatography primarily in the generation of energy during sporulation were purchased from Oriental Yeast (Tokyo). Molecular (13, 14). When operating in the direction of amination of mass standards for SDS-PAGE were purchased from pyruvate to L-alanine, the enzyme can be used as a means Pharmacia LKB. DEAE-Toyopearl 650S, Butyl-Toyopearl of nitrogen assimilation, and it is so used in Streptomyces 650M, and TSK GEL G3000SW column (0.75•~60cm) clavuligerus (15) and Rhodobacter capsulatus (7) since were obtained from Tosoh (Tokyo). Cibacron Blue 3GA was these AlaDHs have relatively low Km values for NH4+. purchased from Sigma. Other reagents of guaranteed grade AlaDH is also found in several cyanobacterial strains were purchased from Wako Pure Chemicals (Osaka). Media and Growth Conditions-P. lapideum, isolated (16) and has been purified and characterized from a N2-fixing cyanobacterium, Anabaena cylindrica (17). In from Matsue hot springs, was grown photoautotrophically Anabaena cells, the enzyme is likely to be less important at 47•Ž as described previously (18). Nitrogen-containing than GS in primary NH4+ assimilation due to its higher Km cultures were grown in medium supplemented with KNO3 value for NH4+, but its importance may increase with (1g/liter) or NH4Cl (1g/liter) for 12 days. Nitrogen increase in the availability of nitrogen (17). starved algae were obtained by bubbling batch cultures However, little information is available about the en which had been growing exponentially in air for 48h zyme from non-N2-fixing cyanobacteria. To study the without nitrogen source. Algal cells for the enzyme purifica tion were grown in medium containing KNO3 (1g/liter), To whom correspondence should be addressed. harvested in the late logarithmic phase by centrifugation Abbreviations: AlaDH, alanine dehydrogenase; PCMB, p-chloro and washed with 200 ml of 0.85% NaCl. The cells were mercuribenzoic acid; DTNB, 5,5•L-dithiobis-(2-nitrobenzoic acid); stored at -20•Ž until use. GluDH, glutamate dehydrogenase; GS, glutamine synthetase; PLP, Enzyme and Protein Assays-The enzyme activity was pyridoxal 5•L-phosphate. Vol. 116, No. 5, 1994 995 996 Y. Sawa et al. assayed at 45•Ž by using a Shimadzu UV240 spectro NH2-Terminal Amino Acid Sequence Analysis-The photometer. purified enzyme was used for the NH2-terminal amino acid Alanine Dehydrogenase (Aminating)-This activity was sequence analysis by automated Edman degradation with a determined by following the oxidation of NADH at 340nm . Shimazu PSQ-1 gas-liquid phase protein sequencer. The standard assay system consisted of 100ƒÊmol of Modification with PLP-The purified enzyme (1ƒÊM) Tris-HCl buffer (pH 8.4), 5ƒÊmol of sodium pyruvate, 133ƒÊ was incubated at 45•Ž with 2 or 5mM of PLP in 50mM mol of ammonium chloride, 0.2ƒÊmol of NADH, and the Tris-HCl (pH 7.2) in a final volume 50ƒÊl. After 20min, the enzyme in a final volume of 1.0ml. The reaction was reaction mixture was added to a freshly prepared solution initiated by the addition of enzyme or substrate and of NaBH4 (final concn., 20mM) to stop the reaction. The followed by measuring the rate of decrease in absorbance at mixture was kept at 45•Ž for 10min and an aliquot (10ƒÊl) 340nm. was used for measurement of the enzyme activity. Alanine Dehydrogenase (Deaminating)-This activity Enzyme Purification-Unless otherwise specified, Tris was determined by following the reduction of NAD. The HCl buffer (pH 7.2) containing 1mM EDTA and 0.05% assay system consisted of 100ƒÊmol of Na2CO3-NaHCO3 2-mercaptoethanol was used as the buffer throughout the buffer (pH 9.2), 10ƒÊmol of L-alanine, 0.2ƒÊmol of NAD, purification. Steps 1 and 2 were carried out at 4•Ž, and and the enzyme in a final volume of 1.0ml. The reaction other steps with the Hitachi 638-30 HPLC system were was initiated by the addition of L-alanine and followed by performed at room temperature. The enzyme activity was measuring the rate of increase in absorbance at 340nm. assayed for the reductive amination of pyruvate. Glutamine Synthetase (Biosynthetic Activity)-This ac Step 1: The washed cells (50g wet weight) were suspend tivity was determined as described previously (18) by ed in 70ml of 0.1M buffer and disrupted for 10min by coupling of the ADP formation with pyruvate kinase and sonication with a 20-kHz KAIJHO DENKI model 300 lactate dehydrogenase reactions. ultrasonic oscillator. The intact cells and debris were Glutamate Dehydrogenase (Aminating)-This activity removed by centrifugation and the supernatant was dia was determined by following the 2-oxoglutarate and ammo lyzed overnight against 20 liters of 25mM buffer. nia-dependent oxidation of NADPH at 340nm according to Step 2: The dialyzed enzyme solution (209ml) was the method of Fisher (19). centrifuged at 30,000•~g for 30min, then applied to a One unit of AlaDH is defined as the amount of enzyme Blue-Sepharose 4B column (5.5•~18cm) equilibrated with that catalyzes the formation of 1ƒÊmol of NAD+ per min in 25mM buffer. The column was washed with the same the reductive amination. The unit definition of GS and buffer until the blue color derived from the phycobilin G1uDH are based on the formation of 1ƒÊmol of product per protein disappeared, then the enzyme was eluted with 25 min. Specific activity is expressed as units per mg of mM buffer containing 0.5mM NAD+. The active fractions protein. Protein was estimated by the method of Lowry et (800ml) were combined and concentrated by ultrafiltration al. (20), with bovine serum albumin as a standard or from through a Toyo UP-20 membrane. the absorption coefficient of the enzyme (A1%1cmat 280nm, Step 3: The enzyme solution was applied to a DEAE 4.37). Toyopearl 650S column (0.8•~25cm) equilibrated with 25 Analytical Electrophoresis-Analytical polyacrylamide mM buffer. The column was washed with the buffer gel electrophoresis was done by the method of Davis (21) in containing 0.1M NaCl, then the enzyme was eluted with a a column of 7.5% polyacrylamide gel with pH 9 buffer linear gradient of 0-0.5M NaCl in 25mM buffer. The system, with a current of 4mA per column. Proteins in the active fractions (4.9ml) were combined and concentrated gel were stained with Coomassie Brilliant Blue (CBB) by Amicon Centricon 30 cartridges. R-250. To find bands with alanine dehydrogenase activity, Step 4: The enzyme solution (0.2ml) was injected onto a gels were also stained with the following mixture: 10mM TSK GEL G3000SW column (0.75•~60cm) equilibrated L-alanine, 0.5mM NAD, 65ƒÊM phenazine methosulfate, with 25mM buffer containing 0.2M NaCl. 35ƒÊM nitroblue tetrazolium, and 100mM Na2CO3 Step 5: The active enzyme fractions (10.4ml) were NaHCO3 buffer, pH 9.2. Gels were incubated for 20min at brought to 20% saturation with ammonium sulfate, then 45•Ž in the dark. SDS-PAGE was performed in 12.5% applied to a Butyl-Toyopearl 650M column (0.4•~15cm) polyacrylamide gel plates by the method of Laemmli (22). equilibrated with 25mM buffer containing 20% saturated Proteins in the gel were stained with CBB R-250. ammonium sulfate.