Phenylalanine Dehydrogenase from Thermophilic Actinomycetes

Vol. 173, No. 13

JOURNAL OF BACTERIOLOGY, JUlY 1991, p. 3943-3948

0021-9193/91/133943-06$02.00/0 Copyright © 1991, American Society for Microbiology

Distribution, Purification, and Characterization of Thermostable Phenylalanine Dehydrogenase from Thermophilic Actinomycetes TOSHIHISA OHSHIMA,l* HARUMI TAKADA,2 TOHRU YOSHIMURA,2 NOBUYOSHI ESAKI,2 AND KENJI SODA2 Department of Chemistry, Kyoto University of Education, Kyoto 612,1 and Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611,2 Japan Received 31 December 1990/Accepted 18 April 1991

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70°C for at least 60 min or in the range of pH 5 to 10.8 at 50°C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.

from Kojin (Tokyo, Japan). Red Sepharose 4B was prepared as previously described (21). Glycine-KCl-KOH buffer was made as described previously (19). [4R-2H]- and [4S-2H] NADH were prepared as previously described (10). All other chemicals were analytical-grade reagents. Microorganisms and growth conditions. Thermophilic microorganisms were obtained from Institute for Fermentation of Osaka, Osaka, Japan, and the German Collection of Microorganisms, Gottingen, Federal Republic of Germany, except for Bacillus acidocaldarius TA 6 and BA 152, which were gifts from Tairo Oshima, Tokyo Institute of Technology, Yokohama, Japan. For determination of enzyme distribution, the microorganisms were grown at 45, 50, or 60°C for 8 to 100 h with rotary shaking in a medium (20 ml) containing 5 g of peptone, 5 g of glycerol, 2 g of yeast extract, 2 g of Ehrlich meat extract, 1 g of KH2PO4, 2 g of K2HPO4 .2H20, 0.1 g of MgSO4 7H20, 0.05 g of CaCl2, 9 FLg of biotin, and 0.4 g of L-tyrosine per liter (pH 7.2 at 25°C). B. acidocaldarius cells were aerobically cultured at 60°C for about 12 h in a medium (pH 3.2) previously described (26). For enzyme purification, T. intermedius IFO 14230 cells were grown at 50°C for 18 h in the same medium except containing 1.5% peptone and 0.01% novobiocin in a 20-liter fermentor (NBS MF-128s-A). Enzyme assay and protein determination. Enzyme activity was assayed spectrophotometrically with a Shimadzu MPS2000 spectrophotometer. The standard reaction mixture for oxidative deamination was composed of 200 ,umol of glycineKCl-KOH buffer (pH 10.85), 10 ,umol of L-phenylalanine, 1.25 j.mol of NAD, and enzyme in a final volume of 1 ml. For reductive amination, the mixture contained 150 ,umol of Na2CO3-NaHCO3 buffer (pH 9.0), 200 ,umol of NH4Cl, 5 ,umol of phenylpyruvate, 0.2 ,umol of NADH, and enzyme in a total volume of 1 ml. The reaction mixture was incubated at 37°C, and the change in absorbance of NADH was monitored at 340 nm. One unit of enzyme is defined as the amount catalyzing the formation of 1 FLmol of NADH per min at 37°C in the oxidative deamination of L-phenylalanine.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) catalyzes the reversible deamination of L-phenylalanine to phenylpyruvate and ammonia in the presence of NAD. It was first found in Brevibacterium sp. isolated from soil (14) and was purified from several mesophiles, Sporosarcina ureae (1, 3), Bacillus sphaericus (3), Bacillus badius (4), Rhodococcus sp. (5, 13), Rhodococcus maris (17), and Nocardia sp. (9), for characterization. The enzyme is important for the synthesis of Lphenylalanine and its related L amino acids from the corresponding oxo analogs and ammonia (2, 12, 13). We have shown that leucine dehydrogenase (EC 1.4.1.9) of a thermophile is much more stable than that of a mesophile not only at high temperatures but also under various other conditions (20). The thermostable leucine dehydrogenase is advantageous to L-leucine production in a membrane reactor (25) and for analysis of substrate amino acids and oxo acids (23). The thermostable enzyme has been studied to elucidate the relationship between structure and catalytic function (18). We first found the thermostable phenylalanine dehydrogenase in a thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230, and showed that it is useful for the selective determination of L-phenylalanine and phenylpyruvate (24). In this report, we describe the distribution of phenylalanine dehydrogenase in thermophiles and the purification and characterization of the enzyme from T. intermedius IFO 14230. MATERIALS AND METHODS Materials. Standard proteins for molecular weight determination were purchased from Sigma Chemical, DEAEToyopearl was purchased from Tosoh (Tokyo, Japan), and Sepharose CL-4B and Sephadex G-100 were purchased from Pharmacia. NAD, NADP, NADH, and NADPH were gifts *

Corresponding author. 3943

3944

OHSHIMA ET AL.

Protein concentration was determined by the method of Lowry et al. (16), with bovine serum albumin as a standard protein. Preparation of crude enzyme solution. Cells washed twice with 0.85% NaCl solution were suspended in 10 mM potassium phosphate buffer (pH 7.2) containing 1 mM EDTA and 0.01% (vol/vol) 2-mercaptoethanol and were disrupted by ultrasonication. The cell debris was removed by centrifugation (20,000 x g, 20 min); the supernatant solution was used as the enzyme solution for screening of phenylalanine dehydrogenase and for purification. Purification of phenylalanine dehydrogenase. Washed cells (160 g [wet weight]) of T. intermedius IFO 14230 were suspended in 300 ml of 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 1 mM EDTA. All procedures were carried out at room temperature (about 20°C) unless otherwise stated. Step I. Heat treatment of crude enzyme solution. The cells were disrupted for 20 min in an ultrasonic oscillator. After centrifugation, solid NaCl was added to the supernatant solution (final concentration, 0.10 M), and then the enzyme solution was heated at 70°C for 1 h. The enzyme was centrifuged at 12,000 x g for 5 min, and the supernatant solution was dialyzed against 1,000 volumes of 10 mM potassium phosphate buffer (pH 6.8) containing 1 mM EDTA and 0.01% 2-mercaptoethanol. Step II. Red Sepharose 4B affinity chromatography. The enzyme solution (400 ml) was applied to five columns (3 by 22 cm) of Red Sepharose 4B equilibrated previously with dialysis buffer (pH 6.8). After each column was washed with the buffer (about 500 ml), the enzyme was eluted with buffer (about 500 ml) supplemented with 0.5 M NaCl. The active fractions were pooled, and then the enzyme (70 ml) was concentrated and deionized by ultrafiltration (Toyo Roshi ultrafilter UP 20 membrane). In this procedure, 10 mM Tris-HCl buffer (pH 8.0) containing 0.01% 2-mercaptoethanol and 1 mM EDTA was substituted for the elution buffer. Step III. DEAE-Toyopearl column chromatography. The enzyme was applied to a DEAE-Toyopearl column (2.5 by 20 cm) equilibrated with 10 mM Tris-HCI buffer (pH 8.0) containing 40 mM NaCl, 0.01% 2-mercaptoethanol, and 1 mM EDTA. The enzyme passed through the column under these conditions. The enzyme collected was concentrated by ultrafiltration. Step IV. Sepharose CL-4B column chromatography. The enzyme solution (about 10 ml) was applied to a Sepharose CL-4B column (1.2 by 100 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing 0.50 M NaCl, 0.01% 2-mercaptoethanol, and 1 mM EDTA. The enzyme was eluted in the fractions corresponding to the void volume of the column and was concentrated. Step V. Sephadex G-100 column chromatography. The enzyme was further purified by Sephadex G-100 chromatography (column, 1.2 by 100 cm) by the procedure described for step IV. Analytical gel electrophoresis. Disc gel electrophoresis (7.5% gel) was carried out as described by Davis (8), and sodium dodecyl sulfate (SDS) slab gel electrophoresis (12% gel, 2 mm thick) was performed by the procedure of Laemmli (15). Molecular weight determination. Molecular weight was determined by gel filtration on a Toyopearl HW-55 column (1.6 by 62 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing 0.1 M NaCl. The following standard proteins were used to make a calibration curve: ferritin (molecular weight, 445,000), B. sphaericus alanine dehydro-

J. BACTERIOL.

TABLE 1. Screening of phenylalanine dehydrogenase in thermophiles Strain

Thermoactinomyces intermedius IFO 14230 ............. T. vulgaris DSM 43354 ........................................ T. vulgaris DSM 43184 ........................................ T. vulgaris DSM 43062 ........................................ T. vulgaris IFO 12516 .......................................... T. vulgaris IFO 13606 .......................................... T. vulgaris IFO 13605 .......................................... T. glaucus IFO 12530 .......................................... Streptomyces thermocastaneus IFO 14275 .............. S. thermoluteus IFO 14269 ................................... Saccharomonospora viridis IFO 12207 .................... Pseudonocardia thermophila IFO 12133 .................. Microbispora bispora DSM 43038 .......................... Thermonospora alba DSM 43310 ...........................

Sp acta (U/mg)

0.123 0.120 0.050 0.029 0.027 0.018 0.012 0.010 0.037 0.015 0.026 0.016 0.013 0.012

a The following microorganisms have specific activities of 0.005 to 0.010 U/mg: Thermoactinomyces vulgaris DSM 43047 and DSM 43016, Thermoactinomyces sacchari DSM 43356, Thermoactinomyces monosporus IFO 14050, Streptomyces thermoviolaceus IFO 12382 and DSM 40443, Streptomyces thermovulgaris IFO 12383 and 13089, Streptomyces thermotolerans IFO 13088, Streptomyces thermoluteus IFO 14270, Streptomyces thermocoerulescens IFO 14273, Streptomyces thermolilacinus IFO 14274, Streptomyces thermocyaneovilaceus IFO 14271, Thermomonosporafusca IFO 14071, Thermomonospora mesouviformis IFO 14178, Micropolyspora cinereoflava IFO 14338, Micropolyspora roseoalba IFO 14337, Micropolyspora faeni DSM 43113, Nocardiodes thermolilacimus IFO 14336, Saccharomonospora viridis DSM 43068, and Bacillus sp. strain 466. Activity was not detected in extracts of the following thermophiles: B. stearothermophilus DSM 297, DSM 456, DSM 457, DSM 458, DSM 1550, DSM 2027, IFO 12550, and IFO 12983; B. sphaericus DSM 461, DSM 462, and DSM 463; B. coagulans DSM 459, DSM 2319, DSM 2320, and DSM 2321; Bacillus sp. strains DSM 405, DSM 406, DSM 411, DSM 465, DSM 730, DSM 1519, DSM 1520, and DSM 1521; B. acidocaldarius TA 6, TA 152, DSM 449, DSM 451, DSM 452, DSM 453, DSM 454, and DSM 455; Thermus aquaticus DSM 625; and Thermus sp. strains DSM 579 and DSM 674.

genase (231,000), lactate dehydrogenase (145,000), bovine serum albumin (67,000), and ovalbumin (43,000). The subunit molecular weight was determined by SDS-polyacrylamide gel electrophoresis with the following marker proteins: bovine serum albumin (molecular weight, 67,000), catalase (57,500), ovalbumin (43,000), ,-lactoglobulin (18,400), and cytochrome c (13,000). Determination of stereospecificity of hydrogen transfer. The stereospecificity of hydrogen transfer of [4R-2H]NADH or [4S-2H]NADH catalyzed by phenylalanine dehydrogenase was studied by the 'H nuclear magnetic resonance (NMR) method (10). [4R-2H]NADH or [4S-2H]NADH (about 5 mM) was incubated with phenylalanine dehydrogenase (0.11 U) in the presence of 5 mM phenylpyruvate and 0.1 M NH3 in 0.6 ml of 2H20. After incubation at 37°C for 3 h, the 'H NMR spectra of the aromatic region of the nicotinamide ring of NAD produced were measured on a 200-MHz NMR apparatus (Varian VXR-200). RESULTS AND DISCUSSION Distribution of phenylalanine dehydrogenase in thermophiles. To find organisms that produce thermostable phenylalanine dehydrogenase, we screened for enzyme activity in various thermophiles from culture collections. As shown in Table 1, high specific activity was found mainly in Thermoactinomyces species; in particular, T. intermedius IFO 14230 and T. vulgaris DSM 43354 and DSM 43184 produced the enzyme abundantly. Enzyme activity was detected in almost all strains of thermophilic actinomycetes tested. The enzyme has been demonstrated in a few strains of mesophiles. Thus,

THERMOSTABLE PHENYLALANINE DEHYDROGENASE

VOL. 173, 1991 TABLE 2. Purification of phenylalanine dehydrogenase from T. intermedius IFO 14230 Step

Total

Crude extract Heat treatment Red Sepharose 4B DEAE-Toyopearl Sepharose CL-4B Sephadex G-100

2,800 2,800 1,680 1,780 661 362

(U/mg)

Yield

(mg)

Sp act

19,500 4,090 220 79 13 4.2

0.144 0.690 7.60 22.4 52.1 86.2

100 100 60 64 24 13

Total

U

protein

-

(%)

3945

(B)

50 (A) 040 30 z 20

0 -

7

o75

3 10

04 L3

3 5

32 u

0 0

0

0.8 0.6 0.2 0.4 Relative mobility FIG. 2. Determination of molecular weights of the native phenylalanine dehydrogenase (A; 0) by gel filtration and of the subunit (B; 0) by SDS electrophoresis. Standard proteins (0) used are described in Materials and Methods. 1.1

1.2

1.3

1.4

Ve / Vo

this is the first example showing the wide distribution of the enzyme in actinomycetes and suggests that the enzyme plays a role in the metabolism of these organisms. Bacillus sp. strain DSM 466 is only the thermophilic Bacillus strain producing phenylalanine dehydrogenase, although only poorly (0.0052 U/mg). T. intermedius IFO 14230 exhibited the highest specific activity of phenylalanine dehydrogenase and produced the enzyme most abundantly. This strain was used for enzyme purification. Addition of L-phenylalanine (5 g/liter), L-tyrosine (0.4 g/liter), or phenylpyruvate (1 g/liter) to the basal medium did not affect enzyme production. The absence of enhancement with L-phenylalanine in enzyme production is peculiar to the T. intermediius IFO 14230 enzyme. Enzyme production in other microorganisms is significantly enhanced by addition of L-phenylalanine to basal medium containing peptone or corn steep liquor (1, 3, 13, 19). Purification of the enzyme. Table 2 summarizes the result of a typical purification of phenylalanine dehydrogenase from the T. intermedius extract. The enzyme was purified about 125-fold with a 13% yield. The purified enzyme was found to be homogeneous by polyacrylamide disc gel electrophoresis (Fig. 1). The activity of this enzyme preparation is higher than that of the preparation obtained in a prelimi-

-

nary study, which probably was partially inactivated during purification (24). Molecular weight and subunit structure. The molecular weight of the phenylalanine dehydrogenase was estimated to be about 270,000 by gel filtration on a Toyopearl HW-55 column (Fig. 2A). The subunit structure was studied by disc gel electrophoresis in the presence of 0.1% SDS. The electrophoresis indicated the presence of only one band, showing a molecular weight of about 41,000 (Fig. 2B). This value is good agreement with that (40,488) calculated from the amino acid sequence of the enzyme (27). Thus, the native enzyme probably has a hexamer structure composed of six identical or similar subunits. The B. sphaericus, S. ureae (3), and B. badius (4) enzymes are octamers, the R. maris enzyme (19) is a dimer, and the Nocardia sp. enzyme (9) is a monomer. This is the first example of the occurrence of a hexameric phenylalanine dehydrogenase. Stability. The thermostability of the enzyme was examined. As shown in Fig. 3A, the enzyme retained its full

_

C

E

a,

(B) (A) FIG. 1. Disc gel electrophoresis of the purified enzyme (about 20 p.g) in the absence of SDS. (A) Activity staining was done at 37°C in a mixture containing 0.1 M Tris-HCl buffer (pH 8.0), 1 mM NAD, 10 mM L-phenylalanine, 0.4 mM phenazine methosulfate, and 0.5 mM p-iodonitrotetrazolium chloride until a band of sufficient intensity was visible. (B) The protein band was stained with Coomassie brilliant blue R-250. The position of the tracking dye was at the bottom of the gel.

FIG. 3. Effects of temperature and pH on enzyme stability. (A) Thermostability. After heat treatment of the pure enzyme in 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 1 mM EDTA, an aliquot of the enzyme solution was withdrawn and the remaining activity was assayed for the oxidative deamination of L-phenylalanine at 37°C. Heat treatment was carried out at 70°C (0) and 75°C (0). (B) Effect of pH on stability. After the enzyme in 50 mM buffer of various pHs was incubated at 50°C (0) and 70°C (0) for 10 min, an aliquot of the enzyme solution was withdrawn and the remaining activity was assayed for the oxidative deamination of L-phenylalanine at 37°C. The buffers (50 mM) used were acetate buffer (pH 4.5 to 6.5), potassium phosphate buffer (pH 6.8 to 8.5 and 10.8 to 11.5), and glycine-KCl-KOH buffer (pH 9 to 11).

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J. BACTERIOL.

TABLE 3. Substrate specificity -1 0

0Substrate

o

Deaminationb L-Phenylalanine

-

L-4-Aminophenylalanine L-Leucine

(0

Concn

(mM) 10

Relative activitya 100

5 10

7.2 3.9

5 10 10 10 10 10 5 10

100 13.9 5.5 5.0 3.3 1.3 0.92 0.74

Aminationc

In

a

8

9

10

11

12

pH FIG. 4. Effect of pH on enzyme activity. The oxidative deamination of L-phenylalanine (0) was determined at different pHs with 0.2 M glycine-KCl-KOH buffer, and the reductive amination of phenylpyruvate (0) was determined with 0.15 M Na2CO3-NaHCO3 buffer and 0.2 M NH4Cl-KOH buffer. The pH of each reaction mixture was measured with a pH meter after the reaction. The maximum activities for the oxidative deamination of L-phenylalanine and the reductive amination of phenylpyruvate were 86 U/mg and 170 ,umol/mg, respectively.

activity upon heating at 70°C for 60 min, but the activity was lost rapidly by heating above 75°C. This feature of thermostability is similar to that of other dehydrogenases from moderate thermophiles such as B. stearothermophilus leucine dehydrogenase (20). The stability of the enzyme at various pHs is shown in Fig. 3B. Activity was not lost upon incubation between pH 5.0 and 10.8 at 50°C for 10 min. The enzyme was stable between pH 5.5 and 9.5 upon heating at 700C for 10 min. The S. ureae and B. sphaericus enzymes were completely inactivated by incubation at 50 and 60°C, respectively, for 10 min (3). Therefore, the T. intermedius enzyme is much more thermostable than the enzymes from mesophiles. This finding suggests the possibility that the thermostable enzyme is more useful than the enzymes from mesophiles as a biocatalyst for L-phenylalanine production (12, 14) and analysis (24). Effect of pH. The enzyme showed maximum reactivity at pH 11.0 for the oxidative deamination of L-phenylalanine. The optimum pH of phenylpyruvate amination was around 9.2 (Fig. 4). The high reactivity of the enzyme at rather high pHs is similar to those of other amino acid dehydrogenases such as leucine dehydrogenase (19, 20) and alanine dehydrogenase

(22).

Substrate specificity. The enzyme shows high specificity for L-phenylalanine in oxidative deamination, and L-tyrosine is inert; the T. intermedius enzyme differs from the enzymes of mesophiles (3, 5, 9) in this respect. As shown in Table 3, only L-4-aminophenylalanine and L-leucine are slightly oxidized by the enzyme. For reductive amination, phenylpyruvate is the preferred substrate. In addition, the enzyme catalyzes the amination of several 2-oxo acids such as

2-oxo-4-methylthiobutyrate, 2-oxo-4-methylpentanoate, and 2-oxo-3-methylbutylate, although slowly (Table 3). The enzyme does not act on 4-hydroxyphenylpyruvate, unlike phenylalanine dehydrogenase from mesophiles (3, 4, 9, 13, 17). The high substrate specificity is favorable for the selective determination of L-phenylalanine and phenylpyruvate (24). Ammonia is an exclusive substrate as the amino donor for reductive amination. Hydroxylamine, methylamine, and ethylamine (0.15 M) were inert as the amino donor. The enzyme required NAD (NADH) as a coenzyme, as

Phenylpyruvate 2-Oxo-4-methylthiobutyrate 2-Oxo-4-methylpentanoate 2-Oxo-4-methylbutyrate DL-2-Oxo-3-methylpentanoate 2-Oxobutyrate Oxaloacetate Pyruvate

a Specific activity on the deamination of L-phenylalanine was 86.2 U/mg, and activity on the reductive deamination of phenylpyruvate was 170 ,umol/ mg. b Inert: L-tyrosine (2.55 mM), L-tryptophan (5 mM), D-phenylalanine (10 mM), DL-phenylglycine (4 mM), DL-homophenylalanine (4 mM), L-methionine (10 mM), DL-tert-leucine (5 mM), L-alanine (50 mM), L-valine (10 mM), L-isoleucine (10 mM), L-histidine (10 mM), L-glutamate (10 mM), L-proline (10 mM), and glycylglycine (10 mM). ' Inert: 4-hydroxyphenylpyruvate (2.5 mM) and 2-oxoglutarate (10 mM).

does the mesophile enzyme, and NADP (NADPH) was inert. Steady-state kinetic mechanism. A series of steady-state kinetic analyses was carried out to identify the reaction mechanism (6, 7, 19). First, initial velocity studies were performed. The double-reciprocal plots of initial velocity (v) against L-phenylalanine concentrations in the presence of several fixed concentrations of NAD gave intersecting straight lines (Fig. 5A). This pattern shows that the reaction proceeds via the formation of a ternary complex of the enzyme with NAD and L-phenylalanine, as with other amino acid dehydrogenases (19, 22). The Km values for NAD and L-phenylalanine were calculated to be 0.078 and 0.22 mM, respectively, from the secondary plots of intercepts versus reciprocal concentrations of the other substrate. Doublereciprocal plots of v against phenylpyruvate concentrations at several fixed concentrations of ammonia and a high and constant concentration of NADH gave straight intersecting lines (Fig. 5B). At a high concentration of phenylpyruvate, the double-reciprocal plots of v against NADH concentrations at several fixed concentrations of ammonia gave also intersecting lines (Fig. 5D). On the other hand, similar plots of v against NADH concentrations at several different concentrations of phenylpyruvate and a high concentration of ammonia gave parallel lines (Fig. 5C). These patterns may rule out the possibility of random addition of the substrates and represent a sequential ordered mechanism in which ammonia binds to the enzyme between other two substrates. The Km values for NADH, phenylpyruvate, and ammonia were determined to be 0.025, 0.045, and 106 mM, respectively. Product inhibition studies of oxidative deamination were performed to determine the order of substrate addition and product release. With NADH as an inhibitor, the doublereciprocal plots of v versus NAD concentrations at a constant concentration of L-phenylalanine showed a typical competitive inhibition pattern (Fig. 6). This finding indicates that NAD and NADH can bind to the free form of the enzyme. NADH showed noncompetitive inhibition against L-phenylalanine. Phenylpyruvate showed noncompetitive

THERMOSTABLE PHENYLALANINE DEHYDROGENASE

VOL. 173, 1991

3947

(A) 2H

Phepyr

H

CONH2

CONH2 R

+

NH3

R

COOH

R

[4-2H] -NAD

[4R-2H] -NADH

CH2 H-C-NH2 L-Phe

(B)

I N H31-1 (mM -1) 1NADHF1(mM-') FIG. 5. Initial velocity analysis. (A) Double-reciprocal plots of v (initial velocity) versus NAD concentration at a series of fixed concentrations of L-phenylalanine: 1.0 (1), 0.4 (2), 0.2 (3), and 0.1 (4) mM. (B) Double-reciprocal plots of v versus phenylpyruvate (Phepyr) concentration at several fixed concentrations of ammonia at a constant NADH concentration (0.10 mM). Ammonia was used at concentrations of 120 (1), 80 (2), 40 (3), and 20 (4) mM. (C) Double-reciprocal plots of v versus NADH concentration at several fixed concentrations of phenylpyruvate at a constant ammonia concentration (200 mM). Phenylpyruvate was used at concentrations of 0.040 (1), 0.020 (2), 0.015 (3), and 0.010 (4) mM. (D) Double-reciprocal plots of v versus ammonia concentration at several fixed concentrations of NADH at a constant phenylpyruvate concentration (10 mM). NADH was used at concentrations of 0.040 (1), 0.020 (2), 0.014 (3), and 0.010 (4) mM.

and uncompetitive inhibitions with respect to phenylalanine and NAD, respectively. Ammonia showed uncompetitive inhibition with respect to both L-phenylalanine and NAD (data not shown). Noncompetitive inhibition by phenylpyruvate with respect to phenylalanine rules out the TheorellChance mechanism (28). These initial velocity and product

4

0.063 0.04 -2

0.02

-

0

10

20

(NADI-1 (mM-1) FIG. 6. Product inhibition by NADH with respect to NAD. Inhibition by NADH was analyzed with NAD as the varied substrate in the presence of L-phenylalanine (5 mM). The NADH concentrations were 0 (1), 0.050 (2), 0.070 (3), and 0.100 (4) mM.

9.2

9.1

9.0

8.9

8.8

8:7

8:6 PPM

FIG. 7. Aromatic region of the 'H NMR spectra of NAD after incubation of [4R-2H]NADH (A) or [4S-2H]NADH (B) with phenylalanine dehydrogenase, ammonia, and phenylpyruvate (Phepyr). Other conditions are given in the text.

inhibition patterns suggest a sequential ordered binaryternary mechanism, in which the sequence of substrate binding to the enzyme in the oxidative deamination is NAD and L-phenylalanine and then the sequence of product release is phenylpyruvate, ammonia, and NADH, but the inhibition by NADH against L-phenylalanine was noncompetitive. Only this feature does not fit with the suggested mechanism. Some unknown factor, now under investigation, is probably involved in the reaction. Stereospecificity of hydrogen transfer. NAD- and NADPdependent dehydrogenases show pro-R or pro-S stereospecificity for hydrogen removal from the C-4 position of the nicotinamide moiety of the reduced coenzymes. We studied the stereospecificity of hydrogen transfer of NADH catalyzed by phenylalanine dehydrogenase by using [4R2H]NADH and [4S-2H]NADH. When [4R-2H]NADH was incubated with phenylalanine dehydrogenase in the presence of ammonia and phenylpyruvate, a resonance doublet around 88.8 for the C-4 proton of NAD was not observed in the 'H NMR spectrum (Fig. 7A). This finding suggests that 2H is present at the C-4 position of NAD. In contrast, the doublet signal for the C-4 proton of NAD was shown in the NMR spectrum after incubation of a similar reaction mixture containing [4S-2H]NADH (Fig. 7B). This result shows that the 4S-2H of NADH was removed and 'H remained at the C-4 position of NAD. Thus, the thermostable phenylalanine dehydrogenase of T. intermedius is pro-S stereospecific, like leucine dehydrogenase (19), glutamate dehydrogenase (29), and B. sphaericus phenylalanine dehydrogenase (3) and unlike alanine dehydrogenase (20) and r-lysine dehydrogenase (11).

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The results presented here show that phenylalanine dehydrogenase of T. intermedius IFO 14230 has a few characteristics distinct from those of mesophiles: it has a hexameric structure, high thermostability, and high substrate specificity. Its high stability and substrate specificity favor its use for amino acid production and analysis (24). ACKNOWLEDGMENTS This work was supported in part by grants from Bioindustry Development Center, Tokyo, and The Agricultural Chemical Foundation of Japan, Tokyo, and by grants-in-aid for scientific research (62616005, 63616005, and 01616005) from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Asano, Y., and A. Nakazawa. 1985. Crystallization of phenylalanine dehydrogenase from Sporosarcina ureae. Agric. Biol. Chem. 49:3631-3632. 2. Asano, Y., and A. Nakazawa. 1987. High yield of L-amino acids by phenylalanine dehydrogenase from Sporosarucina ureae. Agric. Biol. Chem. 51:2035-2036. 3. Asano, Y., A. Nakazawa, and K. Endo. 1987. Novel phenylalanine dehydrogenase from Sporosarucina ureae and Bacillus sphaericus: purification and characterization. J. Biol. Chem. 262:10346-10354. 4. Asano, Y., A. Nakazawa, K. Endo, Y. Hibino, M. Ohmori, N. Numao, and K. Kondo. 1987. Phenylalanine dehydrogenase of Bacillus badius: purification, characterization, and gene cloning. Eur. J. Biochem. 168:153-1597. 5. Campagna, R., and A. F. Buckmann. 1987. Comparison of the production of intracellular L-phenylalanine dehydrogenase from Rhodococcus species M4 and Sporosarcina ureae at 50 liter scale. Appl. Microbiol. Biotechnol. 26:417-421. 6. Cleland, W. W. 1971. Steady state kinetics, p. 1-43. In P. D. Boyer (ed.), The enzymes, 3rd ed., vol. 2. Academic Press, New York. 7. Dalziel, K. 1975. Kinetics and mechanism of nicotinamidenucleotide linked dehydrogenases, p. 1-60. In P. D. Boyer (ed.), The enzymes, 3rd ed., vol. 11A. Academic Press, New York. 8. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404427. 9. de Boer, L., M. van Rijssel, G. J. Euverink, and L. Dijkhuizen. 1989. Purification, characterization and regulation of a monomeric L-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239. Arch. Microbiol. 153:12-18. 10. Esaki, N., H. Shimoi, N. Nakajima, T. Ohshima, H. Tanaka, and K. Soda. 1989. Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenase. J. Biol. Chem. 264: 9750-9752. 11. Hashimoto, H., H. Misono, S. Nagata, and S. Nagasaki. 1989. Stereospecificity of hydrogen transfer of the coenzyme catalyzed by L-lysine r-dehydrogenase. Agric. Biol. Chem. 53:11751178. 12. Hummel, W., E. Schmidt, C. Wandrey, and M.-R. Kula. 1986. L-Phenylalanine dehydrogenase from Brevibacterium sp. for production of L-phenylalanine by reductive amination of phe-

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