J. Biochem. 84, 1133-1138 (1978)
from Klebsiella pneumoniae Shintaro KIKUCHI and Makoto ISfflMOTO Department of Chemical Microbiology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060 Received for publication, March 30, 1978
D-Serine dehydratase [EC 4.2.1.14] was purified from a strain of Klebsiella pneumomae 140fold from crude extract with a yield of 5 %. This enzyme catalyzed the formation of pyruvate and ammonia not only from D-serine but also from L-serine, and also catalyzed the formation of a-ketobutyrate and ammonia from D-threonine. Km values for D-senne, L-serine, and D-threonine were 2.8 ITIM, 20 mM, and 3.6 mM, respectively. Km for pyridoxal 5'-phosphate was 2.5 /JM. The molecular weight was estimated to be 46,000 by Sephadex G-150 gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis. This enzyme was inducible by r>serine. Induction by casamino acids appeared to depend on the presence of D-serine.
It has been considered that most enzymes involved in amino acid metabolism use only one of the optical isomers as a substrate. However, some enzymes have been reported to act upon both optical isomers, though the rates are different. Tryptophan 2,3-dioxygenase [EC 1.13.11.11] from rabbit intestine (7) and glutamine synthetase [EC 6.3.1.2] from sheep brain (2) act not only on the L-isomer but also on the D-isomer. Serine dehydratase, catalyzing the non-oxidative deamination of serine to pyruvate and ammonia, has been known for a long time (5). D-Serine dehydratase [EC 4.2.1.14] was purified from Neurospora crassa (4) and Escherichia coll (5,6) and well-characterized. D-Serine and D-threonine were used as substrates but the L-forms were not used at all. During an investigation on amino acid oxidation in nitrate respiration, the authors found that Abbreviations: BPB, bromphenol blue; Rm, relative mobility; SDS, sodium dodecyl sulfate. Vol. 84, No. 5, 1978
1133
D-serine dehydratase from Klebsiella pneumoniae isolated from soil (7) formed pyruvate not only from D-serine but also from L-serine. The enzyme was purified to a homogeneous state and some properties were investigated. These results are presented in this communication. EXPERIMENTAL PROCEDURE Materials—D-Serine and L-serine were products of Wako; the content of the D-amino acid in L-serine was found to be less than 0.08% (mol/ mol) from the amount of oxygen consumed in the presence of D-amino-acid oxidase [EC 1.4.3.3]. [a^ values of preparations of D-serine, L-serine, and D-threonine were +6.81°, -6.81°, and +28°.0, respectively. D-Amino-acid oxidase of hog kidney was obtained from Sigma (grade "Crude") and from Worthington (code " D A O F F ' ) , and aldolase, bovine serum albumin, ovalbumin, and chymotrypsinogen from Boehringer Mannheim.
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A D-Serine Dehydratase Acting also on L-Serine
1134
ously equilibrated with 0.01 M buffer. Elution was performed stepwise with 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, and 0.5 M buffer. The activity was eluted at 0.2-0.4 M. Assay of D-Serine Dehydratase—The reaction mixture contained 0.1 fimo\ of pyridoxal 5'-phosphate, 50 [imo\ of D-serine, L-senne, or D-threonine, and 40 /imol of phosphate buffer, pH 7.0, in a final volume of 1.0 ml. After incubation at 30°C for 30 min, the reaction was stopped by adding 1.0 ml of 10 % trichloroacetic acid. After removing the precipitate by centrifugation, a-keto acid in the supernatant was determined by the 2,4-dinitrophenylhydrazine method (8), using xylene as an extracting solvent. Enzyme activity is expressed in ftmo\ of ar-keto acid formed per min. Identification of the Reaction Product—This was done by paper chromatography of 2,4-dinitrophenylhydrazones obtained from the reaction mixture. The solvent for development was nbutanol-//-propanol-benzene-6 % NH 4 OH (15 : 50 15 : 20, v/v) (9). Other Assays—Ammonia formed was assayed by the indophenol method (70) after microdiffusion {11). Amino acids were determined by the ninhydrin method (12) after elimination of ammonia by microdiffusion. Protein was determined by Lowry et al. (13) using bovine serum albumin as a standard. D-Serine in the casamino acids medium was estimated by the procedure of Larson et al. (14). Estimation ofMolecular Weight—The molecular weight of D-serine dehydratase was estimated by filtration of the purified preparation (0.4 ml) through a column of Sephadex G-150 gel (1.9 X 110 cm). The standard proteins used were aldolase (molecular weight, 158,000), bovine serum albumin (67,000), ovalbumin (45,000), and chymotrypsinogen (25,000). Electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate (SDS) was also performed with the purified preparation treated with 1 % SDS and 1 mM mercaptoethanol at 37°C for 1 h (15). Standard proteins used were bovine serum albumin, ovalbumin, and chymotrypsinogen. Polyacrylamide Disc Gel Electrophoresis— After electrophoresis (16) and staining with Amido black, the mobility relative to bromphenol blue (BPB) was determined. r>Serine dehydratase activity was located by enzyme assay using extracts of gel slice 1 mm wide. /. Biochem.
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Organism—A strain of K. pneumoniae isolated from soil (7) was used. Cultivation—Culture media contained 10 g of casamino acids (Difco) or 1.5 g of D-serine, 300 fig of biotin, 500 fig of pyridoxine hydrochloride, 500 fig of thiamine hydrochloride, 27.2 g of KH,PO 4 , 28.2 g of K,HPO 4 , 5 ml of salt solution, and 2 g of KNO 3 in 1 liter. Salt solution contained 1 g of MgS(V7H,0, 0.1 g of MnCV4H s O, 40 mg of FeSO 4 -7H,O, 10 mg of CaCl t , and 5 mg of NaiMoO 4 -2HiO in 100 ml, and the pH was adjusted to 2 with H,SO4. In some experiments, casamino acids was replaced by 0.5% glucose and 50 mM NH4C1. A culture of K. pneumoniae grown in the casamino acids medium was transferred to 20 ml of the D-serine medium and incubated at 30°C for 12 h in deep layer culture. Purification of D-Serine Dehydratase Apoenzyme—All procedures were carried out at about 4°C in potassium phosphate buffer, pH 7.0. Cells (90 g wet weight) suspended in 200 ml of 0.1 M buffer were disrupted by sonic disintegration (Tomy UR-150) at 20 kHz, 133 W, for 10 min. The sonicate was centrifuged at 100,000 xg for 120 min and the supernatant was used as crude extract (Sloo). This was subjected to DEAEcellulose column (3 x40 cm) chromatography. The adsorbed protein was eluted with a linear gradient system formed from 1,000 ml of 0.1 M KC1-0.02 M buffer and 1,000 ml of 0.7 M KC1-0.02 M buffer. The eluted fraction containing activity was dialyzed against 0.02 M buffer or diluted with the same buffer and applied to a column of DEAE-celluIose (3 x 25 cm). The adsorbed protein was eluted with a linear gradient system formed from 500 ml of 0.02 M buffer and 500 ml of 0.5 M KC1-0.02 M buffer. The active fraction was concentrated by ultrafiltration with a UK-10 filter (Toyo Roshi) to 3 ml and passed through a column of Sephadex G-150 (1.9x110 cm). The active fraction of the eluate was dialyzed against 0.02 M buffer and applied to a column of DEAE-Sephadex A-25 (2x16 cm). Elution was performed with a linear gradient system formed from 200 ml of 0.02 M buffer and 200 ml of 0.5 M KC1-0.02 M buffer containing 10 fiM pyridoxal 5'-phosphate. The active eluted fraction was dialyzed against distilled water or diluted with the same volume and applied to a column of hydroxyapatite (1.5x5 cm) previ-
S. K1KUCHI and M. ISHIMOTO
D-SERINE DEHYDRATASE FROM K. pneumonias
1135
RESULTS AND DISCUSSION
TABLE I. Summary of the purification of D-serine dehydratase.
Purification step
Crude extract (S1Oo) 1st DEAE-cellulose 2nd DEAE-cellulose Sephadex G-150 DEAE-Sephadex A-25 Hydroxyapatite
Total protein (mg)
Activity for D-serine Total (units)
5,100
460
992
397
180
320
102
200
7.0
51
2.1
26
Specific (units/mg protein) 0.09 0.40 1.78 1.96 7.3 12
Activity for L-serine Specific (units/mg pro tern)
Yield
(%)
Total (units)
(100)
11.9
0.002
(100)
86
9.3
69
8.0
43
4.4
11
1.2
0.009 0.044 0.043 0.17 0.29
Yield
5.6
0.6
(%)
78 67 37 10 5.3
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Purification of D-Serine Dehydratase, and Its Substrate Specificity—D-Serine dehydratase was purified from cells of K. pneumoniae grown on D-serine by two DEAE-celluose, Sephadex G-150, DEAE-Sephadex A-25, and hydroxyapatite chromatographies. The process of purification is summarized in Table I. The purified preparation had specific activity 140 times that of the crude extract. The specific activity for L-serine was 1/40 of that for r>serine and the ratio did not change during purification; the yield was 5% in both cases.
The peak of activity for L-serine coincided with that for D-serine in each chromatogram. The purified preparation gave a single protein band in disc gel electrophoresis with a relative mobility of 0.64 (Fig. 1). The enzymatic activities were found to have the same relative mobilities; 0.63 for D-serine, 0.64 for L-serine, and 0.63 for D-threonine. The findings indicate that D-serine dehydratase purified to a homogeneous state also acted on L-serine and D-threonine. The relative activity of the purified preparation in forming keto acids from several amino acids (20 nun) is listed in Table LL D-Threonine was decomposed more rapidly than L-serine, but L-threonine • was not
TABLE n. Substrate specificity of D-serine dehydratase. Experimental details are given in the text. The amount of pyruvate formed from D-senne was 5.75 ftmol. Exp. No.
Fig. 1. Polyacrylamide disc gel electrophoresis of purified D-serine dehydratase. Experimental details are given in " EXPERIMENTAL PROCEDURE." Vol. 84, No. 5, 1978
1 2 3 4 5 6 7 8 9 10 11
Substrate D-serine L-serine D-threonine L-threonine D-alanine L-alanine D-cysteine L-cysteine D-serine+D-alanine D-serine+L-threonine D-seri ne+D-cysteine
a-keto acid formed • (7o)
100 3.1 14 0 0 0 0 0 94 99 93
1136
20 40 60 60 INCUBATION TIME(mln) Fig. 2. Stoichiomctry of D-scnne dehydratase. The reaction mixture contained 40 DIM phosphate buffer, pH 7.0, 0.1 mM pyridoxal S'-phosphate, 10 RIM D-serine and the purified preparation in a final volume of 1.0 ml. Incubation was carried out at 30°C. (A) D-serine disappearance and pyruvate formation; O, D-serine and • , pyruvate. (B) D-senne disappearance and NH, + formation; O, D-serine and • , NH, + .
(0.40) or L-serine (0.41) and D-threonine (0.13), respectively. pH Optimum and Km—The optimum of Dserine decomposition was found to lie at pH 6.57.5 (Fig. 3). Km values for D-serine, L-serine, and D-threonine were 2.8 mM, 20 mM, and 3.6 mM, respectively. Pyridoxal 5'-phosphate was indispensable for the reaction and had the effect of stabilizing the enzyme; Km was 2.5 /UM. Estimation of Molecular Weight—The molecular weight of D-serine dehydratase was determined by Sephadex G-150 gel filtration to be 46,000 from plots of the logarithm of molecular weight versus elution volume. The molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 40,000. As this value is not very different from the value obtained by gel filtration, the enzyme protein appears to have no subunit structure. Comparison of K. pneumoniae D-Serine Dehydratase with the Same Enzyme from Other Organisms —Table III shows some properties of D-serine dehydratase from various organisms. The molecular weight of the K. pneumoniae enzyme is not different from those of others, though the pH optimum was a little lower. It is characteristic of the present enzyme that L-serine is used as a substrate in addition to D-serine or D-threonine. Induction of D-Serine Dehydratase under Various Growth Conditions—Since K. pneumoniae did not utilize serine as a main carbon source under anaerobic conditions in the absence of nitrate, dehydratase activity for D- and L-serine was assayed in crude extracts from cells grown under aerobic conditions or under anaerobic conditions in the presence of nitrate (Table IV). D-Serine
7.0
8jD
PH
Fig. 3. Effect of pH on D-serine dehydratase. Phosphate buffer; O, Tris-HCI buffer. J. Biochem.
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affected, D- and L-alanine, as well as D- and Lcysteine were not utilized as substrates and did not inhibit the formation of pyruvate from D-serine. D-Serine decreased at an almost constant rate and eventually disappeared completely. The amounts of pyruvate and ammonia formed were equal to the amount of r>serine lost throughout the reaction, as shown in Fig. 2. The reaction products were identified by paper chromatography of the 2,4-dinitrophenylhydrazones as pyruvate (Rf 0.41) and a-ketobutyrate (0.13) from D-serine
S. KIKUCHI and M. ISHIMOTO
D-SERINE DEHYDRATASE FROM K. pneumoniae
1137
TABLE III. The companson of properties of D-serine dehydratase from various organisms.
N. crassa
PH optimum
D-serine (0.29 mM)
8.1-8.2
D-threonine E. coli W
D-senne (1.3 mM) D-threonine (3.2 mM)
E. coli K-12
Reference
D-serine (0.7 mM)
An rwi HU, KAnJ
7. 3-8.0
(5)
17 inn
8.0
(ff)
D-threonine (0.91 mM) K. pneumoniae
D-serine (2.8 mM)
AC ruvu
HO,
D-threonine (3.6 mM)
IMT
6. 5-7.5
40,000»
L-serine (20 mM) » Molecular weight was estimated by the gel filtration method. acrylamide gel electrophoresis.
b
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Molecular weight
Substrate specificity (Km)
Organism
Molecular weight was estimated by SDS-poly-
TABLE IV. Induction of D-senne dehydratase. Growth medium contained: 1, 0.5% glucose and 50mM NH4C1; 2, 10 mM D-serine; 3, 10 ITIM L-senne; 4, 1% (w/v) casamino acids as a growth substrates. KNOa, 20 mM, was added in expenments under anaerobic conditions. Cultures were incubated for 12 h under an N, atmosphere (anaerobic conditions) or with shaking by a rotary shaker in air (aerobic conditions). Activities forming pyruvate from D- and L-senne were assayed in the crude extract from the grown cells. Specific activity of cell extract for Exp. No
1 2 3 4
Growth substrate
Glucose-NH4C1 r>Serine L-Senne Casamino acid
Growth condition
anaerobic anaerobic aerobic anaerobic aerobic anaerobic aerobic
(+KNO,) (+KNOJ
0.03 98
(+KNO,)
31
9.3 9.0
(+KNO,)
— 9.7
90
dehydratase was induced by D-serine under both conditions. When L-serine was used as a growth substrate, the activity was only 1/10 of that of cells grown in the presence of D-serine, though the growth was not different. When cells were grown anaerobically in casamino acids medium in the presence of nitrate, the crude extract obtained had a rather high specific activity. This seems to be due to the presence of D-serine in the casamino acids medium; Vol. 84, No. 5, 1978
D-serine L-senne (munits/mg protein)
36 4.7
7.0 10
6.0 —
the amount of D-serine was estimated to be 0.2 mM by amino acid analysis before and after treatment with D-amino-acid oxidase (14). REFERENCES
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S. KIKUCHI and M. ISHIMOTO