J. Biochem. 116, 995-1000 (1994)

Purification and Characterization of 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 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+ , 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, ; 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 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 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. After the column had been washed with Determination of Molecular Weight-Molecular mass of the same buffer, the enzyme was eluted with a linear the AlaDH subunit was estimated by SDS-PAGE on 12.5% gradient of 20-0% saturated ammonium sulfate in the gel. The proteins used as molecular mass standards were a buffer. The active fractions (12.8ml) were pooled and mixture of ƒ¿-lactalbumin (Mr 14,400), soybean trypsin concentrated by use of Amicon Centricon-30 cartridges. inhibitor (Mr 20,100), carbonic anhydrase (Mr 30,000), The concentrated AlaDH solution was extensively dialyzed ovalbumin (Mr 43,000), bovine serum albumin (Mr against the buffer and stored at 4•Ž. The enzyme was stable, 67,000), and phosphorylase b (Mr 94,000). Molecular mass showing no loss of activity after 2 months of storage. of the native AlaDH was estimated by HPLC on a TSK GEL G3000SW column (0.75•~60cm), equilibrated with 25 RESULTS mM Tris-HCl (pH 7.2) containing 0.2M NaCl at a flow rate of 0.5ml per min. The column was calibrated with the Purification of AlaDH from Phormidium lapideum-We following proteins: horse heart cytochrome c (Mr 12,400), have purified for the first time to electrophoretic homoge yeast adenylate kinase (Mr 32,000), yeast enolase (Mr neity the alanine dehydrogenase of a cyanobacterium. The 67,000), pig heart lactate dehydrogenase (Mr 142,000), and results of a typical enzyme purification are shown in Table yeast GluDH (Mr 290,000). I. A 989-fold purification was achieved, with a 13% recov

J. Biochem. Alanine Dehydrogenase from P. lapideum 997

ery and a final specific activity of 183 units/mg. Analytical aminating reaction was found at 60-70•Ž. The enzyme was disc gel electrophoresis of the purified enzyme gave a single stable at up to 50•Ž upon incubation for 10min at pH 8.4 protein band which corresponded to that staining for the (Fig. 2). However, in the presence of NADH (0.3mM), the activity (data not shown). enzyme stability increased, with retention of 70% of the Molecular Mass and Subunit Structure -The native original activity after the treatment at 60•Ž for 10min molecular mass was determined to be 240,000 Da by gel (Fig. 2). Other substrates showed no protective effect. filtration on a TSK G3000SW column. SDS-PAGE reveal Substrate Specificity-The relative rates of reaction with ed a single protein band of molecular mass 41,000 Da on the NAD(H), NADP(H), 2-oxo acids, and amino acids are basis of its mobility relative to those of standard proteins. shown in Table II. Although it specifically uses NAD(H) as Thus, the enzyme appears to be a hexamer composed of six opposed to NADP(H), AlaDH was less specific with regard identical subunits. to analogs of pyruvate. It showed 15.4 of the control NH2-Terminal Amino Acid Sequence-The NH2-termi activity with 10mM of 3-hydroxypyruvate. Of the amino nal amino acid sequence (21 amino acid residues) of the acids tested, only L-alanine was a substrate for oxidative enzyme was determined by automated Edman degradation: deamination reaction of AlaDH. Apparent Km values were Met-Glu-lle-Gly-Val-Pro-Lys-Glu-Ile-Lys-Asn-Gln-Glu-Phe determined under standard assay conditions for oxidative - Arg-Val-Gly-Leu-Ser-Pro-Ser. deamination and reductive-amination reactions and ob Effect of pH on Alanine Dehydrogenase Activity-The tained from the secondary plots of intercepts versus recip effect of pH on AlaDH activity is shown in Fig. 1. The rocal concentrations of substrates. The Km values for enzyme appeared to be stable throughout the pH range of 6 L-alanine, NAD+, pyruvate, NH4+, and NADH were 5.0, 9. The pH optimum was 8.4 for the reductive amination 0.04, 0.33, 111, and 0.02mM, respectively, at the optimum reaction and 9.2 for the oxidative deamination reaction. At pH of the individual reaction. The Km value for NH4+ the optimum pH for each reaction, the amination rate was varied from 60-200mM depending on the pH, being lowest nine times higher than the deamination rate. at high pH levels (pH 8.6 or above). Thermal Properties-When the enzyme was assayed at Effecters-The inhibitory effect of various compounds on various temperatures, the maximum activity for the the activity for reductive amination was examined by assaying the enzyme in a reaction mixture (Table III). The enzyme was inhibited strongly by Hg2+, p-chloromercuri benzoate, and DTNB, typical inhibitors of SH-, but this inhibition was relieved by 2-mercaptoethanol. 2-Mercaptoethanol alone activated the enzyme. Ions such as Cd2+, Cu2+, and Zn2+ were also inhibitory, but EDTA and

Fig. 1. The effect of pH on enzyme stability (A) and activity (B). (A) The enzyme in 100mM buffer of various pHs was treated at 50•Ž for 5min, then the remaining enzyme activity was assayed for the reductive amination of pyruvate. The 100mM buffers were acetate (pH 4.0-6.5, •›), potassium phosphate (pH 6.0-8.0, •¢), Tris/ HCl (pH 7.5-9.0, •œ), and carbonate (pH 8.5-10.0, •£). (B) The enzyme activity was assayed in buffers of various pHs for the Fig. 2. The effect of temperature on enzyme activity. After reductive amination of pyruvate (solid line) and the oxidative incubation (•›: no addition, •œ: +0.3mM NADH) at various tempera deamination of L-alanine (dashed line). The 100mM buffers used ture for 10min, the remaining enzyme activity was assayed for the were potassium phosphate (pH 6.3-7.6, • ), Tris/HCl (pH 7.0-8.8, reductive amination of pyruvate at 45•Ž. •› •œ), and carbonate (pH 8.8-9.6, •¢ •£).

TART.F, 1. Purification of alanine dehydrogenase from Phormidium lapideum.

Vol. 116, No. 5, 1994 998 Y . Sawa et al.

TABLE II. Substrate specificity of alanine dehydrogenase TABLE IV. The effect of various amino acids on the activity of from Phormidium lapideum. Inert: D-alanine, L-aspartate , DL-2 alanine dehydrogenase from Phormidium lapideum. The en - amino-butyrate, L-serine, glycine, L-glutamate . Activities are ex zyme activity was assayed for the reductive amination of pyruvate. pressed as a percent of that obtained with the primary substrates No inhibition: L-cysteine, L-aspartate, L-arginine, L-leucine, L NADH, NAD+, pyruvate, , and L-alanine which was approx -isoleucine, L-valine, L-histidine, L-lysine. imately 180ƒÊmol NADH oxidized-min-1.mg protein-1 in the ami nating direction and 20ƒÊmol NAD+ reduced-min-1•Emg protein-1 in the deaminating direction.

TABLE III. The effect of various compounds on the activity of alanine dehydrogenase from Phormidium lapideum. The en zyme activity was assayed for the reductive amination of pyruvate. TABLE V. The effect of PLP, and copresence of PLP and the substrate on the enzyme activity. After modification of PLP as described under "MATERIALS AND METHODS," the enzyme activity was assayed for the reductive amination of pyruvate.

a The enzyme was incubated with 5mM PLP and the various com pounds as described under "MATERIALS AND METHODS." b The concentration of NADH and pyruvate were 2 and 10mM, respectively.

TABLE VI. The activities of alanine dehydrogenase, glu tamate dehydrogenase, and glutamine synthetase in NO3 - grown, NH4+-grown, and nitrogen-starved cultures of Phor midium lapideum.

a Mercaptoethanol. aSpecific activity.

,ƒ¿•L-dipyridyl had no effect on the activity. Cibacron Blue Variation of AlaDH Activities with Culture Condi inhibited AlaDH activity and the inhibition mode was tions-The specific activities of AlaDH, GluDH, and GS, competitive with respect to NADH (K1=1.0ƒÊM) and assayed in crude extracts of Phormidium lapideum grown noncompetitive with respect to pyruvate. Of L-amino acids with various nitrogen sources are shown in Table VI. tested, alanine and serine (possibly because of its structural GluDH, which operates for amination in many organisms at similarity to alanine) markedly inhibited the aminating high NH4+ concentration, shows some activity in NO activity of the enzyme; threonine, phenylalanine, and 3- grown and NH4+-grown cultures. GS had the highest tryptophan were also somewhat inhibitory (Table IV). activity regardless culture conditions. In comparison with PLP Modification-When incubated with PLP followed NO3-grown culture, AlaDH activity increased 1.7-fold in by reduction with sodium borohydride, the enzyme was nitrogen-starved culture, similarly to GS activity, and inactivated considerably (Table V). The presence of both 1.2-fold in NH4+-grown culture. GluDH activity was en

pyruvate and NADH largely protected the enzyme against hanced in NH4+-medium and repressed considerably under this inactivation. NADH also considerably protected the nitrogen starvation. enzyme, whereas pyruvate alone protected it only a little and L-alanine or NAD+ alone offered little protective effect.

J. Biochem.

ƒ¿ Alanine Dehydrogenase from P. lapideum 999

DISCUSSION AlaDHs hitherto purified from various microorganisms include monomers, tetramers , hexamers, and an octamer, as described by Ohshima et al. (8) . The enzyme from a non-N2-fixing filamentous cyanobacterium , P. lapideum, Fig. 3. Comparison of the N-terminal amino acid sequence of appears to be composed of six subunits of M, 41,000. Li AlaDH from Phormidium lapideum with those of the enzymes near alignment of the NH2-terminal amino acid se from other sources. N-terminal sequences of the B. subtilis enzyme quences of the Phormidium AlaDH and other AlaDHs and the M. tuberculosis enzyme were cited from Refs. 12 and 11. Those of the B. stearothermophilus enzyme and the B. sphaericus revealed marked similarity (Fig. 3) . The homologies enzyme were cited from Ref. 10. Identical residues are shown by between the Phormidium AlaDH and three Bacillus en asterisks. Gaps (hyphens) were inserted for maximum matching. zymes showed 62-71% identity and that between Phor midium and Mycobacterium enzymes showed 48% identity . The homologies calculated considering conservative (7), and Anabaena cylindrica (17), and its role in the changes were 87-90%. Since all these enzymes are hex regulation of amino acid metabolism has been discussed. amers, the sequence homologies might be high . On the However, the inhibition of the Phormidium AlaDH by other hand, FASTA homology search (National Institute of L-amino acids does not seem to play a significant role in the Genetics, Mishima) through PIR (Protein Identification enzyme regulation, since the intracellular concentrations of Resource: release 39.0, 12/93) database revealed that the these effecters, as determined by HPLC, are considerably NH,-terminal amino acid sequences of AlaDHs showed no lower than those required to affect the enzyme activity in similarities with other amino acid dehhaminoaciddehydrogenases. vitro (data not shown). The Phormidium enzyme has an absolute substrate The Phormidium enzyme was inhibited severely by the specificity for L-alanine and NAD+. In this respect, it modification with PLP, similarly to various other dehy resembles the enzymes from Streptomyces species (6, 24) drogenases including leucine dehydrogenase (27), GluDH and Anabaena variabilis (17) but differs from those from (28), and lactate dehydrogenase (29). B. sphaericus AlaDH Bacillus species (1, 23), Pseudomonas (4), and Rhodo was not affected at all by PLP, as previously reported (1), bacter (7). Absolute specificity for pyruvate in the direction although the enzyme has a consensus sequence (GGK) of reductive amination has not yet been described for any corresponding to Lys80, which was modified by PLP in the enzyme. Although 3-hydroxypyruvate was also a good leucine dehydrogenase (30). Interestingly, the correspond substrate for the Phormidium enzyme, similar to the ing sequence of the Mycobacterium AlaDH was not GGK Anabaena enzyme, 2-oxoglutarate was not a substrate, in but GGR (11). We recently cloned and sequenced the contrast to the Anabaena enzyme (17). The Phormidium Phormidium gene coding for AlaDH: the corresponding enzyme had apparent Km values for substrates other than sequence of the Phormidium AlaDH was GGR, the same as NH4+ that were of the same magnitude as those of AlaDHs that of the Mycobacterium enzyme (Sawa et al., unpub from other species. However, the Phormidium enzyme had lished results). The site of modification by PLP will there a high Km value (111mM) for NH4+. Although the Km value fore be different from that of leucine dehydrogenase. for NH4+ was variable in the range of 60-200mM depend AlaDHs have been observed in several different organ ing on the pH, similar to those of the Anabaena (17) and isms and in different roles. The physiological role of AlaDH Bradhyrhizobium enzymes (25), even the lowest Km value in non-N2-fixing cyanobacteria has not yet been studied in for NH4+ (60.6mM at pH 8.7) was higher than the reported detail, but the following points suggest the physiological values of others. A Km value of this magnitude would role of the Phormidium AlaDH. First, GluDH operating for certainly make amination inefficient at the intracellular amination in many organisms at high NH4+ concentration NH4+ level. In general, hexamer AlaDHs so far reported shows some activity in NO3- and NH4+-grown cultures of P. have a high Km value for NH4+ (16-300mM) compared to lapideum. Second, although the Phormidium AlaDH shows those (4.7-20mM) of tetramer or octamer enzymes. good aminating activity, it is less active than GS in Free thiol groups seem to be essential for the activity of NO3-grown cells of P. lapideum (Table VI). Third, since the AlaDH, since Hg2+ ions, PCMB, and DTNB inhibited the optimum pH for oxidative deamination by Phormidium enzyme activity, and this inhibition was relieved by 2 AlaDH is nearer neutral (9.2) than that of other AlaDHs, - mercaptoethanol. Although two AlaDHs from Bacillus which have high pH optima (10.0-11.2), the enzyme may species were reported to be inhibited severely by -SH operate in oxidative deamination in the cell. Fourth, the Km reagents (3, 8), linear alignment analysis of the overall for NH4+ of AlaDH, although highly variable depending on amino acid sequence revealed no conserved cysteine resi the pH, is higher than those of other AlaDHs and about due (data not shown). These findings suggest that the 200-fold higher than that of Phormidium GS (18). Thus, we blocked cysteine residue may be in a conformationally strongly suggest that the AlaDH in P. lapideum cells plays sensitive position as well as in a position sensitive to a role in deamination, unlike the enzyme from a N2-fixing oxidation. This blocked cysteine residue of AlaDHs may be cyanobacterium, Anabaena variabilis. an residue but not a catalytic residue. We are currently studying the primary structure of the The AlaDH aminating activity of P. lapideum was inhib enzyme derived from the DNA sequence to elucidate the ited in vitro by alanine, serine, threonine, and aromatic relationship between structure and function of the enzyme. amino acids. Similar inhibition has been found in AlaDHs of Details of gene cloning and DNA sequencing for the Streptomyces species (6, 15, 26), Pseudomonas sp. (4), Phormidium AlaDH will be reported. Bradhyrhizobium japonicum (25), Rhodobacter capsulatus

Vol. 116, No. 5, 1994 1000 Y. Sawa et al.

drogenase of the ƒÀ-lactam antibiotic producer Streptomyces clavuligerus. Arch. Microbiol. 125, 137-142 REFERENCES 16. Neilson, A.H. and Doudoroff, M. (1973) Ammonia assimilation in 1. Ohshima, T. and Soda, K. (1979) Purification and properties of blue-green algae. Arch. Microbiol. 89, 15-22 17. Rowell, P. and Stewart, W.D.P. (1976) Alanine dehydrogenase of alanine dehydrogenase from Bacillus sphaericus. Eur. J. Bio the N2-fixing blue-green alga, Anabaena cylindrica. Arch. chem. 100, 29-39 2. Itoh, N. and Morikawa, R. (1983) Crystallization and properties Microbiol. 107, 115-124 18. Sawa, Y., Ochiai, H., Yoshida, K., Tanizawa, K., Tanaka, H., and of L-alanine dehydrogenase from Streptomyces phaeochromo Soda, K. (1988) Glutamine synthetase from a cyanobacterium, genes. Agric. Biol. Chem. 47, 2511-2519 Phormidium lapideum: Purification, characterization, and com 3. Yoshida, A. and Freese, E. (1965) Enzymic properties of alanine dehydrogenase of Bacillus subtilis. Biochim. Biophys. Acta 96, parison with other cyanobacterial enzymes. J. Biochem. 104, 917-923 248-262 19. Fisher, H.F. (1985) L-Glutamate dehydrogenase from bovine 4. Bellion, E. and Tan, F. (1987) An NAD+-dependent alanine liver in Methods in Enzymology (Meister, A., ed.) Vol. 113, pp. dehydrogenase from a methylotrophic bacterium. Biochem. J. 16-27, Academic Press, New York 244,565-570 20. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 5. Porumb, H., Vancea, D., Muresan, L., Presecan, E., Lascu, I., Petrescu, I., Porumb, T., Pop, R., andBarzu, O. (1987) Structural (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 and catalytic properties of L-alanine dehydrogenase from Bacil 21. Davis, B.J. (1964) Disc electrophoresis 2. Method and applica lus cereus. J. Biol. Chem. 262, 4610-4615 tion to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404 6. Vancura, A., Vancurova, I., Volc, J., Jones, S.K.T., Fileger, M., - 427 Basarova, G., and Behal, V. (1989) Alanine dehydrogenase from 22. Laemmli, U.K. (1970) Cleavage of structural proteins during the Streptomyces fradiae. Eur. J. Biochem. 179, 221-227 assembly of the head of bacteriophage T4. Nature 227, 680-685 7. Caballero, F.J., Cardenas, J., and Castillo, F. (1989) Purification 23. Sakamoto, Y., Nagata, S., Esaki, N., Tanaka, H., and Soda, K. and properties of L-alanine dehydrogenase of the phototrophic (1990) Gene cloning, purification and characterization of ther bacterium Rhodobacter capsulatus E1F1. J. Bacteriol. 171, mostable alanine dehydrogenase of Bacillus stearothermophilus. 3205-3210 J. Ferment. Bioeng. 69, 154-158 8. Ohshima, T., Sakane, M., Yamazaki, T., and Soda, K. (1990) 24. Vancurova, I., Vancura, A., Volc, J., Neuzil, J., Flieger, M., Thermostable alanine dehydrogenase from thermophilic Bacillus Basarova, G., and Behal, V. (1988) Purification and partial sphaericus DSM 462. Eur. J. Biochem. 191, 715-720 characterization of alanine dehydrogenase from Streptomyces 9. Smith, M.T. and Emerich, S.W. (1993) Alanine dehydrogenase aureofaciens. Arch. Microbiol. 150, 438-440 from soybean nodule bacteroids: Purification and properties. 25. Muller, P. and Werner, D. (1982) Alanine dehydrogenase from Arch. Biochem. Biophys. 304, 379-385 bacteroids and free living cells of Rhizobium japonicum. Z. 10. Kuroda, S., Tanizawa, K., Sakamoto, Y., Tanaka, H., and Soda, Naturforsch. 37c, 927-936 K. (1990) Alanine dehydrogenases from two Bacillus species 26. Vancurova, I., Vancura, A., Vole, J., Neuzil, J., and Behal, V. with distinct thermostabilities: Molecular cloning, DNA and (1989) A further characterization of alanine dehydrogenase from protein sequence determination, and structural comparison with Streptomyces aureofaciens. J. Basic Microbiol. 29, 185-189 other NAD(P)+-dependent dehydrogenases. Biochemistry 29, 27. Ohshima, T., Misono, H., and Soda, K. (1978) Properties of 1009-1015 crystalline leucine dehydrogenase from Bacillus sphaericus. J. 11. Andersen, A.B., Andersen, P., and Ljunggvist, L. (1992) Struc Biol. Chem. 253, 5719-5725 ture and function of a 40,000-molecular-weight protein antigen of 28. Smith, E.L., Austen, B.M., and Nyc, J.F. (1975) Glutamate Mycobacterium tuberculosis. Infect. Immun. 60, 2317-2323 dehydrogenases in The Enzymes (Boyer, P.D., ed.) 3rd ed., Vol. 12. Siranosian, K.J., Ireton, K., and Grossman, A.D. (1993) Alanine 11, pp. 293-367, Academic Press, New York dehydrogenase (ald) is required for normal sporulation in 29. Chen, S.-S. and Engel, P.C. (1975) Dogfish M4 lactate dehy Bacillus subtilis. J. Bacteriol. 175, 6789-6796 drogenase-Reversible inactivation by pyridoxal 5•L-phosphate 13. McCowen, S.M. and Phibbs, P.V., Jr. (1974) Regulation of and complete protection in complexes that mimic active ternary alanine dehydrogenase in Bacillus licheniformis. J. Bacteriol. complex. Biochem. J. 151, 447-449 118,590-597 30. Matsuyama, T., Soda, K., Fukui, T., and Tanizawa, K. (1992) 14. Frankel, A.D. and Jones, R.F. (1980) Changes in enzyme activity Leucine dehydrogenase from Bacillus stearothermophilus: Iden during differentiation in Chlamydomonas reinhardtii. Biochim. tification of active-site lysine by modification with pyridoxal Biophys. Acta 630, 157-164 phosphate. J. Biochem. 112, 258-265 15. Aharonowitz, Y. and Friedrich, C.G. (1980) Alanine dehy

J. Biochem.