Purification, properties, and partial amino acid sequences of alanine racemase from the muscle of the black tiger prawn Penaeusmonodon

Comparative Biochemistry and Physiology Part B 133 (2002) 445–453

Purification, properties, and partial amino acid sequences of alanine racemase from the muscle of the black tiger prawn Penaeus monodon Naoko Yoshikawaa, Naoshi Dhomaeb, Koji Takiob, Hiroki Abea,* a

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan b The Institute of Physical and Chemical Research, Wako, Saitama 350-0198, Japan Received 10 July 2002; received in revised form 11 September 2002; accepted 12 September 2002

Abstract Alanine racemase wEC 5.1.1.1x, which catalyzes the interconversion between D- and L-alanine, was purified to homogeneity from the muscle of black tiger prawn Penaeus monodon. The isolated enzyme had a molecular mass of 44 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 90 kDa on gel filtration, indicating a dimeric nature of the enzyme. The enzyme was highly specific to D- and L-alanine and did not catalyze the racemization of other amino acids. Km values toward both D- and L-alanine were almost equal and considerably high compared with those of bacterial enzymes. The purified enzyme retained its activity in the absence of pyridoxal 59-phosphate as a cofactor but carbonyl reagents inhibited the activity, suggesting the tightly binding of the cofactor to the enzyme protein. Several partial amino acid sequences of peptide fragments of the purified enzyme showed positive homologies from 52 to 76% with bacterial counterparts and a catalytic tyrosine residue of the bacterial enzyme was also retained in the prawn one, indicating alanine racemase gene is well conserved from bacteria to invertebrates. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: monodon

D-Alanine;

Alanine racemase;

D-Amino

acid; Amino acid sequence; Enzymatic properties; Isolation; Prawn; Penaeus

1. Introduction Free D-amino acids, previously convinced to exist exclusively in eubacteria, have also been clarified to distribute from lower invertebrates ¨ ¨ (Schottler et al., 1984; Portner et al., 1986; Low et al., 1996) to mammals (Hashimoto et al., 1993; Nagata et al., 1994; Kera et al., 1995; Hamase et al., 1999). In a wide range of animal kingdom, several aquatic animals such as crustaceans (D’An*Corresponding author. Tel.: q81-3-5841-5296; fax: q813-5841-8166. E-mail address: [email protected] (H. Abe).

iello and Giuditta, 1980; Okuma and Abe, 1994a,b; Okuma et al., 1995) and bivalve mollusks (Matsushima et al., 1984; Felbeck and Wiley, 1987; Yamada and Matsushima, 1992; Okuma et al., 1998) have been known to contain an extraordinarily copious amount of free D-alanine up to 50 mM in their several tissues. D-Alanine together with L-form has been clarified to be a major osmolyte for intracellular isosmotic regulation both in crustaceans (Okuma and Abe, 1994a; Abe et al., 1999a,b) and bivalves (Matsushima et al., 1984; Yamada and Matsushima, 1992; Okuma et al., 1998). It has also recently been proposed that

1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 1 8 7 - 2

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N. Yoshikawa et al. / Comparative Biochemistry and Physiology Part B 133 (2002) 445–453

D-alanine works as one of the anaerobic end products during anaerobiosis of crayfish Procambarus clarkii (Fujimori and Abe, 2002) and hard clam Meretrix lusoria (Okuma et al., 1998). This function of D-alanine has been predicted since over two decades ago for some invertebrates such as ¨ annelid and sipunculid (Schottler et al., 1984; ¨ Portner et al., 1986). In these crustaceans and mollusks, it has been proven that D-alanine is synthesized from L-alanine by alanine racemase wEC 5.1.1.1x catalyzing the interconversion between these enantiomers (Matsushima et al., 1984; Matsushima and Hayashi, 1992; Fujita et al., 1997a). Invertebrate alanine racemase was partially purified at first from the muscle of black tiger prawn Penaeus monodon (Fujita et al., 1997b), and recently purified to homogeneity from the tail muscle of crayfish (Shibata et al., 2000), mantle muscle of a clam Corbicula japonica (Nomura et al., 2001) and hepatopancreas of P. monodon (Uo et al., 2001). For obtaining a clue to unveil the physiological function and metabolic regulation of free D-alanine in invertebrates, structural and functional investigations on invertebrate alanine racemase are required. However, the structural and enzymatic properties of the enzyme have not yet been clarified in detail for eukaryotes. The above-mentioned isolation trials failed to get an enough amount of enzyme protein to analyze primary structure of the enzyme, partly because of the instability of invertebrate enzyme compared with the bacterial counterpart and a small amount of the enzyme in invertebrate tissues. Since the amino acid sequence of bacterial enzyme is not helpful for analyzing cDNA of invertebrate alanine racemase, it is necessary to get an enough amount of the purified enzyme from invertebrate tissues and determine its partial amino acid sequences for designing primers. In the present study, we report the purification of alanine racemase from the muscle of P. monodon to homogeneity and several enzymatic properties. Also reported are the partial amino acid sequences of the isolated enzyme and the homology between the bacterial and prawn sequences.

2. Materials and methods 2.1. Materials Frozen black tiger prawn P. monodon cultured in Sri Lanka were obtained from the Tokyo Central

Wholesale Fish Market. The tail muscle of the prawn was used for the enzyme purification. DEAE- and Butyl-Toyopearl 650 M and TSK G3000 SWXL were obtained from Tosoh (Tokyo, Japan). Phenyl-Sepharose, HiLoad 16y60 Superdex 200 and Mono Q columns were from Pharmacia (Uppsala, Sweden). Bio-Scale Ceramic Hydroxyapatite Type I column (CHT2-I) was purchased from Bio-Rad Laboratories (Hercules, CA). Sumichiral OA-5000, Aquapore RP-300 and Mightycil RP-18 columns were purchased from Sumika Chemical Analysis Service (Osaka, Japan), Applied Biosystems (Forster, CA) and Kanto Reagents (Tokyo, Japan), respectively. A centrifugal concentrator, Centriprep YM-10, was from Millipore Corporation (Bedford, MA). Molecular weight markers were obtained from Roche Diagnostics (Mannheim, Germany). All other reagents were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO) or Wako Pure Chemical Industries (Osaka, Japan). 2.2. Enzyme and protein assays Enzyme activity was assayed by determining Dand L-alanine with high-performance liquid chromatography (HPLC). Reaction mixture contained 0.1 M Tris–HCl buffer (pH 8.5), 200 mM D- or L-alanine and enzyme solution in a final volume of 1 ml. After 10–30 min incubation at 37 8C, a 0.1-ml aliquot was taken and deproteinized with 0.1 ml of 0.6 M perchloric acid. After centrifugation at 24 000=g for 2 min, the supernatant was neutralized with 0.1 ml of 0.6 M potassium bicarbonate and centrifuged as above. The resulting supernatant was injected into HPLC for D- and Lalanine determination. Alanine enantiomers were separated by an HPLC system (Jasco; Tokyo, Japan) equipped with a chiral column, Sumichiral OA-5000 (f4.6=150 mm). As the mobile phase, 1-mM copper sulfate was used at a flow rate of 1 mlymin. At an ambient temperature, D- and L-alanine eluted from the column were monitored at 254 nm as alanine– copper complex. Enzyme activity was calculated from the increase of D- or L-alanine and expressed as mmolyminØmg protein. Protein concentration was determined according to the method of Lowry et al. (1951) or by a Bio-Rad protein assay kit in the final purification step, with bovine serum albumin as a standard.

N. Yoshikawa et al. / Comparative Biochemistry and Physiology Part B 133 (2002) 445–453

2.3. Purification of alanine racemase Step 1: The muscle of P. monodon, weighing 800 g, was homogenized with 3 volumes of 10 mM Tris–HCl buffer (pH 8.0) containing 50 mM KCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 20 mM pyridoxal 59-phosphate (PLP). The homogenate was centrifuged at 4000=g for 50 min and the supernatant was obtained. All procedures were carried out at 4 8C. Step 2: The supernatant was fractionated with ammonium sulfate (30–70% saturation). The precipitate was dissolved into a minimal volume of 0.1 M Tris–HCl buffer containing 1 mM EDTA, 0.2 mM PMSF and 20 mM PLP, and dialyzed overnight against the same buffer. The dialyzate was centrifuged at 24 000=g for 20 min. Step 3: The supernatant was applied onto a DEAE-Toyopearl column (f5=25 cm) equilibrated with 0.1 M Tris–HCl buffer (pH 8.0) containing 20 mM PLP (buffer A). The enzyme was eluted with a linear gradient of 0–0.25 M NaCl in buffer A and the active fractions were pooled and brought to 1 M solution with ammonium sulfate. Step 4: The enzyme solution was applied to a Butyl-Toyopearl column (f4=25 cm) equilibrated with buffer A containing 1 M ammonium sulfate. The enzyme was eluted with a linear gradient of 1–0 M ammonium sulfate in buffer A and active fractions were collected. Step 5: The enzyme solution was directly applied onto a Phenyl-Sepharose column (f2.5=15 cm) equilibrated with 10 mM Tris– HCl buffer (pH 8.0) containing 20 mM PLP (buffer B) and 0.5 M ammonium sulfate. The enzyme was eluted with a linear gradient of 0.5– 0 M ammonium sulfate in buffer B. The active fractions were pooled, concentrated and washed with 10 mM potassium phosphate buffer (pH 8.0) containing 20 mM PLP (buffer C) using a Centriprep YM-10. Step 6: The above active fraction was loaded onto a CHT2-I column (f7=52 mm) previously equilibrated with buffer C. The enzyme was eluted with the same buffer at a flow rate of 0.5 mlymin. The active fractions were concentrated by Mono Q column (f5=50 mm) equilibrated with buffer C at a flow rate of 1 mlymin. The enzyme was eluted with a step gradient of 1 M NaCl in buffer C. Step 7: The enzyme solution was applied to a Superdex 200 column (f1.6=60 cm) equilibrated

447

with buffer B containing 0.15 M NaCl. The active fraction was collected as the final preparation. 2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) in 10% slab gel. The gel was stained with Coomassie Brilliant Blue R-250. 2.5. Amino acid sequence analysis For amino acid sequence analysis, the enzyme was reduced with DTT and pyridylethylated overnight with 4-vinylpyridine in 0.5 M Tris–HCl (pH 8.5) containing 10 mM EDTA and 7 M guanidine hydrochloride in the dark at room temperature. The reaction mixture was applied on a gel permeation HPLC with two tandem columns of TSK G3000 SWXL (7.8=300 mm2 each) in 6 M guanidine hydrochloride containing 10 mM sodium phosphate (pH 6.0) using a Gilson model 302 pump (Villiers-le-Bel, France) and a Hewlett Packard HP 1040 M diode-array detection system (Fort Collins, CO). Peptides were eluted with 0.09% (vyv) aqueous trifluoroacetic acid (TFA) and subsequently 80% (vyv) acetonitrile containing 0.075% (vyv) TFA, and monitored by the absorbance at 215, 254, 275 and 290 nm. The pyridylethylated enzyme was fragmented chemically or enzymatically by the following two methods. Cleavage with cyanogen bromide was carried out in 70% formic acid at room temperature and digestion with Achromobacter protease I was carried out in 0.5 M Tris–HCl (pH 8.5) containing 8.0 M urea and 1 mM EDTA at 37 8C overnight. The peptide fragments obtained from the above two methods were separated by RP-HPLC using a Gilson HPLC system equipped with an Aquapore RP-300 (f2.1=30 mm) or a Mightycil RP-18 column (f2=50 mm). The molecular masses of the peptide fragments were determined by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry with a reflex mass spectrometer (Bruker; Bremen, Germany) using 2-benzothiazolethiol as the matrix. Amino acid sequence analysis was carried out on a Model 477A protein sequencer connected on line to a Model 120A PTH analyzer (Applied Biosystems).

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3. Results and discussion 3.1. Purification Alanine racemase was purified from the muscle of P. monodon to homogeneity by ammonium sulfate fractionation and successive DEAE-Toyopearl, Butyl-Toyopearl, Phenyl-Sepharose, CHT2I and Superdex 200 column chromatographies. The overall purification was approximately 127 592fold with 16% yield (Table 1). Specific activity of the final preparation was 2807 mmolymin mg protein. In all chromatographies, the enzyme eluted as a single peak as previously shown in the partial purification (Fujita et al., 1997b), suggesting the low possibility of multiple forms of the enzyme in prawn muscle. Unlike microbial enzymes, the prawn enzyme was rather unstable. In the purification procedure, the enzyme gradually lost its activity in the solution of ammonium sulfate or NaCl. In addition to the instability, the enzyme was small in amount in the prawn muscle, approximately 0.45 mgyg muscle calculated from the final amount of protein obtained and the yield shown in Table 1 and an initial 800-g muscle. From these reasons, it was difficult to obtain an enough amount of enzyme protein for its structure analysis. This was the largest problem in the purification and characterization of invertebrate alanine racemase. However, we hereby could get an enough amount of protein (57 mg) for structural analysis. The molecular mass of the purified enzyme was estimated by SDS-PAGE (Fig. 1). The enzyme gave a single band with a molecular mass of approximately 44 kDa, which was almost identical to that of the hepatopancreas enzyme from the same prawn (41 kDa; Uo et al., 2001). Silver staining of the final preparation on SDS-PAGE

Fig. 1. SDS-PAGE analysis of purified alanine racemase. Protein was stained with Coomassie Brilliant Blue R-250. Lane 1, pooled active fractions after DEAE-Toyopearl column chromatography; lane 2, Butyl-Toyopearl; lane 3, Phenyl-Sepharose; lane 4, CHT-I; lane 5, purified alanine racemase after Superdex 200 column; lane 6, molecular markers, myosin (200 kDa), b-galactosidase (116 kDa), albumin (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa) and myoglobin (17 kDa).

also showed no additional band (data not shown). An apparent molecular mass of the enzyme in its native form was determined to be approximately 90 kDa by gel filtration using a Superdex 200 column (Fig. 2). These data indicate that alanine racemase exists as a dimer in prawn muscle as is the case in the hepatopancreas enzyme (80 kDa; Uo et al., 2001). Bacterial alanine racemases have been reported to be a monomer or dimer having an identical subunit with a molecular mass of approximately 40 kDa (Yoshimura and Soda,

Table 1 Purification of an alanine racemase from the muscle of black tiger prawn Penaeus monodon Purification step

Protein (mg)

Total activity (mmolymin)

Yield (%)

Specific activity (mmolyminØmg)

Purificationa (fold)

Step Step Step Step Step Step Step

44 823 24 523 1468 147 2.79 0.76 0.057

976 861 770 552 405 293 160

100 88 79 57 42 30 16

0.022 0.035 0.525 3.755 145.2 384.2 2807

1 1.6 23.9 171 6598 17 464 127 592

a

1 2 3 4 5 6 7

Crude extract 30–70% (NH4)2SO4 DEAE-Toyopearl Butyl-Toyopearl Phenyl-Sepharose CHT2-I Superdex 200

Ratio of the specific activity at each step to that of crude extract.

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Fig. 2. Determination of molecular mass of alanine racemase with Superdex 200 column chromatography. Molecular markers are aldolase (158 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa) and cytochrome c (12 kDa).

1994). The bivalve enzyme has also been reported to have a subunit molecular mass of 41 kDa but a native molecular mass of 140 kDa, suggesting the trimeric or tetrameric nature of the bivalve enzyme (Nomura et al., 2001). Thus, the molecular size of the monomer is almost identical for all enzymes thus far isolated from both prokaryotes and eukaryotes. One exception is the enzyme from crayfish

muscle, which has been reported to be a monomeric enzyme with a molecular mass of 58 kDa (Shibata et al., 2000). 3.2. Enzymatic properties From Lineweaver–Burk plots (Fig. 3), the apparent Km values of the enzyme toward L- and D-alanine were calculated to be 167 and 179 mM, respectively (Table 2). Eukaryotic alanine racemase has been reported to have rather high Km values. Km values for L- and D-alanine are 38 and 2 mM for the fungal enzyme from Tolypocladium niveum (Hoffmann et al., 1994), 22.6 and 9.2 mM for molluscan enzyme from C. japonica (Nomura et al., 2001), 171 and 73.5 mM for crayfish enzyme (Shibata et al., 2000), and 150 and 24 mM for prawn hepatopancreas enzyme (Uo et al., 2001), respectively. Thus, the crustacean enzymes show extraordinarily high Km values. Of these Table 2 Kinetic parameters of alanine racemase isolated from the muscle of black tiger prawn Penaeus monodon

Fig. 3. Lineweaver–Burk plots for the racemization of D- and L-alanine. Enzyme activities for both L to D (d) and D to L (s) directions were determined.

Direction

Vmax (mmolyminØmg)

Km (mM)

Kcat (sy1)

KcatyKm (sy1ymM)

L™D

3502 3155

167 179

2568 2314

15.38 12.93

D™L

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eukaryotic enzymes, the muscle enzyme from P. monodon showed the highest Km values which were at least 10-times higher than the D- and Lalanine concentrations in prawn muscle (Okuma et al., 1995). However, the Vmax values and turnover numbers (Kcat) of the prawn enzyme were very high and almost the same for both directions. Thus, the catalytic efficiency (Kcat yKm) was also not different for both directions. The equilibrium constant (Keq) was calculated to be 0.84 from the Haldane equation (Briggs and Haldane, 1925), (Kcat yKm for D-alanine)y(Kcat yKm for L-alanine), and the value was below the theoretical one for racemization (i.e. 1.0). From these data, it is considered that the prawn alanine racemase catalyzes the equilibrium between D- and L-alanine efficiently, in spite of the low content in muscle and high Km values. This is in accord with the ratio of D-y L-alanine in crustacean muscle. In all crustacean muscle thus far examined, the ratio falls in 0.47–0.84 (Okuma et al., 1995) and increases from 0.62 to 0.92 during seawater acclimation of crayfish from 0 to 75% seawater (Fujimori and Abe, 2002). Thus, the enzyme may well regulate the intracellular concentrations of D- and L-alanine in crustaceans inhabiting the salinity fluctuating water. The optimal pH of the enzyme was approximately 9.5 in the direction of L to D and approximately 10 for the reverse direction (Fig. 4). Enzyme activity was maximal at approximately 37 8C for both directions. These data were consistent with those of the other eukaryotic enzymes (Hoff-

Table 3 Effects of some effectors on the activity of alanine racemase Compound

None Pyridoxal 59-phosphate

Concentration (mM)

20

Relative activity (%) L™D

D™L

100 108

100 124

Aminooxyacetate

10 100 1000

37.1 7.95 0

53.8 33.1 0

Hydroxylamine

10 100 1000

51.9 5.64 0

83.6 20.1 0

mann et al., 1994; Shibata et al., 2000; Nomura et al., 2001; Uo et al., 2001). The purified enzyme was highly specific to D- and L-alanine and demonstrated no activity on L-serine, L-glutamate, Laspartate, L-leucine, L-arginine or L-valine as substrate. All known alanine racemases require PLP as a cofactor to form a Schiff base with the substrate (Yoshimura and Soda, 1994). The purified prawn enzyme was slightly activated by the addition of 20 mM PLP, whereas the enzyme showed activity even in the absence of PLP (Table 3). The activity also remained without PLP during the purification procedure. Absorption at 420 nm, corresponding to the Schiff base complex of PLP with an active site lysine (Inagaki et al., 1986; Watanabe et al., 1999), was undetectable in the purified enzyme because of the scarce amount of the enzyme

Fig. 4. Optimal pH and temperature of alanine racemase. (a) Optimal pH. The activity was assayed in 0.1 M potassium phosphate, pH 7.0–7.5, 0.1 M Tris–HCl, pH 8.0–9.0, or 0.1 M CAPS-NaOH, pH 9.5–11.0. (b) Optimal temperature. The reaction time was 30 min at each temperature. Enzyme activities for both L to D (d) and D to L (s) directions were determined.

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Fig. 5. Comparison of partial amino acid sequences of alanine racemase from the muscle of P. monodon to the corresponding region of that from bacteria. Amino acids identical with those of P. monodon are underlined. A catalytic tyrosine residue, which is known as tyrosine 265 in alanine racemase from B. stearothermophilus, is boxed.

protein. By contrast, the enzyme was strongly inhibited by hydroxylamine and aminooxyacetic acid that were known to be the strong inhibitors of PLP-dependent enzymes (Table 3). These data may suggest that PLP binds strongly to the enzyme protein, probably with a covalent bond as is shown in alanine racemase of Bacillus stearothermophilus, where PLP is covalently linked via an aldimine linkage to lysine 39 and other residues support the positioning of PLP in the protein (Shaw et al., 1997). 3.3. Partial amino acid sequences We obtained seven peptide fragments from the purified alanine racemase. Of these fragments, three peptides represented homologies to bacterial alanine racemases when compared using Blast search (Fig. 5), while others showed no significant similarity. One of the homological peptides showed comparatively high positive homology to bacterial alanine racemases. The similarities of this fragment against the bacterial enzymes were calculated to be 58% for Lactobacillus plantarum, 60% for L. reuteri, 57% for Bacillus psychrosaccharolyticus, 53% for B. stearothermophilus, and 52% for Listeria monocytogenes. Alanine racemase of B. stearothermophilus has been reported to have two catalytic residues, lysine 39 which removes the ahydrogen of D-alanine and tyrosine 265 which acts on L-alanine (Shaw et al., 1997; Stamper et al., 1998; Watanabe et al., 1999). As seen in Fig. 5,

this tyrosine residue and several residues around the tyrosine were well conserved in all bacterial alanine racemases and even in the prawn enzyme. Thus, the data suggest that the purified enzyme from P. monodon has an analogous catalytic site with the bacterial alanine racemases and catalyzes the racemization with a similar manner to the bacterial enzymes. This also indicates alanine racemase gene is conserved from bacteria at least to P. monodon during a long evolutional time scale. The second peptide fragment was homologous to near C-terminal regions of alanine racemases from Bacillus psychrosaccharolyticus (75%) and B. subtilis (76%). The third fragment was homologous to putative alanine racemase from Vibrio cholerae (53%) and serine racemase from Enterococcus gallinarum (53%). Unfortunately, N-terminal amino acid sequence could not be identified in the present study in spite of every possible effort. N-terminus of the prawn enzyme is possible to be modified, although 15 residues of N-terminal region have been identified in the crayfish enzyme (Shibata et al., 2000). We are now conducting cDNA cloning of the prawn alanine racemase on the basis of these amino acid sequences. Acknowledgments This work was partly supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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