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Characterization of Nα-benzyloxycarbonyl-l -lysine oxidizing enzyme from Rhodococcus sp. AIU Z-35-1
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 3, 218–223. 2007 DOI: 10.1263/jbb.104.218
© 2007, The Society for Biotechnology, Japan
Characterization of N α-Benzyloxycarbonyl-L-Lysine Oxidizing Enzyme from Rhodococcus sp. AIU Z-35-1 Kimiyasu Isobe1* and Shouko Nagasawa1 Department of Agro-bioscience, Faculty of Agriculture, Iwate University, 3 Ueda, Morioka 020-8550, Japan1 Received 19 April 2007/Accepted 23 June 2007
An oxidase catalyzing conversion of N α-benzyloxycarbonyl-L-lysine (N α-Z-L-lysine) to N α-benzyloxycarbonyl-L-aminoadipate-δ-semialdehyde (N α-Z-L-AASA) was purified from Rhodococcus sp. AIU Z-35-1, and its properties were revealed. This enzyme catalyzed an oxidative deamination of the ε-amino group of N α-acyl-L-lysine and the α-amino group of N ε-acyl-L-lysine. The apparent Km value for N α-acetyl-L-lysine was much larger than that for N ε-acetyl-L-lysine. The peptidyl L-lysines, L-lysine and many other L-amino acids were also oxidized, but N α-acyl-D-lysine, N ε-acylD-lysine and D-amino acids were not. Thus, the conversion of N α-Z-L-lysine into N α-Z-L-AASA was catalyzed by the L-amino acid oxidase with broad substrate specificity. This enzyme, a flavoprotein with a molecular mass of 100 kDa, consisted of two identical subunits of 51 kDa. [Key words: N α-benzyloxycarbonyl-L-lysine, N α-benzyloxycarbonyl-L-aminoadipate-δ-semialdehyde, N α-benzyloxycarbonyl-L-aminoadipic acid, L-amino acid oxidase]
Recently, we isolated a new bacterial strain, named Rhodococcus sp. AIU Z-35-1, for production of N α-benzyloxycarbonyl-L-aminoadipic acid (N α-Z-L-AAA), and a new biochemical method for N α-Z-L-AAA production has been developed using this isolated strain (1). Since this strain converted N α-benzyloxycarbonyl-L-lysine (N α-Z-L-lysine) to N α-Z-L-AAA via N α-benzyloxycarbonyl-L-aminoadipateδ-semialdehyde (N α-Z-L-AASA), it was also useful for the production of N α-Z-L-AASA (2). Although these new bacterial methods were superior to chemical and other biochemical methods in the conversion yields and formation speed of N α-Z-L-AASA and N α-Z-L-AAA, the enzymes catalyzing the conversion of N α-Z-L-lysine to N α-Z-L-AAA have not been identified. We therefore investigated the enzymes catalyzing this conversion, and found an enzyme exhibiting oxidase activity on N α-Z-L-lysine. The present paper describes production, purification and some remarkable properties of the N α-Z-L-lysine oxidizing enzyme produced by Rhodococcus sp. AIU Z-35-1.
Sanyo Fine (Osaka). Peroxidase was gift from Amano Enzyme (Nagoya). All other chemicals used were of analytical grade and commercially available. Cultivation of strain Z-35-1 Rhodococcus sp. Z-35-1 was first incubated in test tube containing 5 ml of N α-Z-L-lysine medium, pH 7.0, consisting of 0.2% KH2PO4, 0.1% Na2HPO4, 0.05% MgSO4 ⋅ 7H2O, 0.5% glucose, and 0.5% N α-Z-L-lysine at 30°C for 2 d with shaking (120 strokes/min). The culture (1.5 ml) was then inoculated into 500 ml shaker flask containing 150 ml of the N α-ZL-lysine medium, and it was cultivated at 30°C for 2 d with shaking. This strain was also cultivated in the L-lysine medium, which consisted of the same as N α-Z-L-lysine medium except that N α-ZL-lysine was replaced by L-lysine, under the same conditions as the N α-Z-L-lysine medium. Then, 20 ml of the second culture was transferred into a 3-l of culture flask containing 2 l of the L-lysine medium. After the third culture was carried out at 30°C for 1 d, the cells were harvested by centrifugation at 20,000 ×g for 10 min, washed with 50 mM potassium phosphate buffer, pH 7.0, and then stored at −20°C until use. Analysis of oxidase activity Oxidase activity was assayed by measuring the formation rate of hydrogen peroxide as follows. The standard reaction mixture contained 40 µmol of N α-Z-L-lysine, 0.6 µmol of 4-aminoantipyrine, 1.94 µmol of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt dihydrate, 6.7 units of peroxidase, 0.1 mmol of potassium phosphate, pH 7.0, and an appropriate amount of enzyme, in a final volume of 1.0 ml. The formation of hydrogen peroxide was spectrophotometrically followed at 30°C for 5 min by measuring the absorbance at 555 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one micromole of hydrogen peroxide per min. Assay of dehydrogenase activity Dehydrogenase activity was assayed by measuring the formation rate of NAD(P)H as follows. The standard reaction mixture contained 40 µmol of Nα-ZL-lysine, 0.6 µmol of β-NAD+ or β-NADP+, 0.37 µmol of nitro
MATERIALS AND METHODS Chemicals L-Lysine and other L-amino acids were purchased from Wako Pure Chemicals (Osaka). N α-Z-L-lysine and N α-Z-D-lysine were purchased from Calbiochem-Novabiochem (Läufelfingen, Switzerland) and Fluka Chemie (Buchs, Switzerland), respectively. Peptidyl L-lysines were obtained from Sigma Chemical Japan (Tokyo). N α-Acetyl-L-lysine, N ε-acetyl-L-lysine and N α-acetyl-D-lysine were from Watanabe Chemical Industries (Hiroshima). N α-ZL-glutamine, N α-Z-L-arginine and N α-Z-L-asparagine were from * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)19-621-6155 218
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blue tetrazolium, 0.2 unit of diaphorase, 0.1 mmol of potassium phosphate, pH 7.0, 0.05% Triton X-100 and an appropriate amount of enzyme, in a final volume of 1.0 ml. The formation of NAD(P)H was spectrophotometrically followed at 30°C by measuring the absorbance at 550 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one micromole of NAD(P)H per min. Purification of enzyme Purification of enzyme was carried out using cells obtained from 24 l culture broth of the L-lysine medium. All procedures were done at 5–10°C. The buffer used was potassium phosphate buffer, pH 7.0. Preparation of cell-free extract Cells (wet weight, 14.7 g) were suspended in 250 ml of 10 mM buffer, and disrupted by a Multi-beads shocker (Yasui Kikai, Osaka) at 2500 rpm for 8 min (2 min × 4). The supernatant (250 ml) was obtained by centrifugation at 20,000 ×g for 10 min. Ammonium sulfate fractionation Solid ammonium sulfate was added to 50% saturation in the supernatant, and resulting precipitate was discarded by centrifugation at 20,000 ×g for 10 min. First Phenyl-Toyopearl column chromatography (stepwise elution) A Phenyl-Toyopearl resin equilibrated with 10 mM buffer containing 1.5 M ammonium sulfate was added into the supernatant, and its suspension was stirred for 1 h. Using this suspension, the column (16 × 2.2 cm diameter) was prepared, and undesirable proteins were washed out with 10 mM buffer containing 1.5 M ammonium sulfate. The oxidase was then eluted with 10 mM buffer containing 0.8 M ammonium sulfate. The active fractions were collected, and ammonium sulfate was added to1.5 M. Second Phenyl-Toyopearl column chromatography The eluate from the first Phenyl-Toyopearl column chromatography was applied again to a Phenyl-Toyopearl column (16 ×2.2 cm diameter) equilibrated with 10 mM buffer containing 1.5 M ammonium sulfate. After the column was washed with the same buffer, the adsorbed enzyme was eluted by a linear gradient with 10 mM buffer containing 1.5 M ammonium sulfate and 0.8 M ammonium sulfate (300 ml each), followed with 150 ml of 10 mM buffer containing 0.8 M ammonium sulfate. The active fractions were collected, and deionized to the conductivity of 1.8 ms/cm by ultrafiltration. DEAE-Toyopearl column chromatography The deionized enzyme solution was applied to a DEAE-Toyopearl column (16 ×1.8 cm diameter) equilibrated with 10 mM buffer, and the column was washed with 10 mM buffer. The adsorbed enzyme was then eluted by a linear gradient with 10 mM buffer and 0.15 M NaCl (200 ml each). The active fractions were collected, and dialyzed against 5 mM buffer. Hydroxyapatite column chromatography The dialyzed enzyme solution was applied to a Hydroxyapatite column (13 ×1.0 cm diameter) equilibrated with 10 mM buffer, and the column was washed with the same buffer. The adsorbed enzyme was then eluted by a linear gradient with 10 mM buffer and 100 mM buffer (50 ml each), followed with 20 ml of 100 mM buffer. The active fractions were collected, and the purity was analyzed. Protein measurement Protein concentration was measured with a Protein Quantification Kit (Dojindo Laboratories, Tokyo). SDS–PAGE and molecular mass SDS–PAGE was performed according to the method of Laemmli (3). Proteins were stained with Coomassie Brilliant Blue R-250. Molecular mass was estimated by gel filtration on a TSK gel G3000SWXL column and by SDS–PAGE using standard markers of molecular mass (Sigma Japan). Isoelectric point The isoelectric point was determined with an isoelectric focusing apparatus (Nippon Eido, Tokyo) under conditions of 1% Ampholine, pH 3.5–10, with a sucrose gradient at 400 V for 2 d at 4°C. One-milliliter fractions were collected, and the pH was measured at 4°C. NH2-terminal amino acid sequence The amino acid sequence
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of the intact enzyme was determined using Applied Biosystems gas-phase protein sequencer equipped with an on-line reversephase chromatography system for identification of PTH-amino acids.
RESULTS Identification of enzyme Enzyme catalyzing conversion of N α-Z-L-lysine into N α-Z-L-AASA was analyzed using cell-free extract, which was prepared from cells incubated for 36 h in the N α-Z-L-lysine medium. The oxidase activity on N α-Z-L-lysine was obtained, but the dehydrogenase activity on N α-Z-L-lysine was less than 2% of the oxidase activity (Table 1). In addition, formation of equimolecular amounts of NH3 and H2O2 was confirmed (data not shown). It was therefore concluded that the conversion of N α-Z-L-lysine into N α-Z-L-AASA was catalyzed by an oxidase according to Fig. 1. Effects of nitrogen source on enzyme production Rhodococcus sp. AIU Z-35-1 was incubated at 30°C for 36 h in the medium containing N α-Z-L-lysine, L-lysine, n-butylamine, ammonium nitrate or ammonium sulfate as a nitrogen source. This strain grew well in the medium containing Nα-Z-L-lysine, L-lysine, ammonium nitrate or ammonium sulfate, and the N α-Z-L-lysine oxidase activity was obtained by culturing with N α-Z-L-lysine or L-lysine, but not with the other compounds tested (Table 2). In addition, the oxidase activity obtained from the L-lysine medium was four times higher than that of the Nα-Z-L-lysine medium. The L-lysine medium was therefore suitable for enzyme production. Purification and molecular mass The purification procedure is summarized in Table 3. The oxidase was purified to an electrophoretically homogeneous state by approximately 155-fold. The purified enzyme showed a single protein band on native- and SDS–PAGE (Fig. 2). The molecular mass of the native and denatured enzymes was estimated to be 100 kDa on TSK gel G3000SWXL column and 51 kDa on SDS–PAGE, respectively (data not shown). These results indicate that this enzyme consisted of two identical subTABLE 1. Identification of enzyme catalyzing conversion of N α-Z-L-lysine to N α-Z-L-AASA Enzyme activity (m unit/100 ml broth) Dehydrogenase Oxidase NADP+ NAD+ α N -Z-L-lysine 13.0 0.21 0 Rhodococcus sp. AIU Z-35-1 was cultured at 30°C for 36 h in the N α-Z-L-lysine medium. Cells of 100 ml culture broth were disrupted with Multi-beads shocker at 2500 rpm for 4 min, and cell debris was discarded by centrifugation at 20,000 × g for 10 min. Enzyme activity was assayed using supernatant under standard assay conditions. Substrate
FIG. 1. Conversion of N α-Z-L-lysine into N α-Z-L-AASA by L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1.
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TABLE 2. Effects of nitrogen source on enzyme production Oxidase activity Protein Specific activity (m unit/100 ml ⋅broth) (mg/100 ml ⋅broth) (m unit/mg ⋅ protein) 15 15.8 0.95 N α-Z-L-lysine L-Lysine 60 21.4 2.80 n-Butylamine 1> 0.27 0 Ammonium sulfate 1> 58.2 0 Ammonium nitrate 1> 17.2 0 Rhodococcus sp. AIU Z-35-1 was incubated at 30°C for 36 h in the medium containing indicated chemicals as a nitrogen source. N α-Z-L-lysine oxidase activity was assayed using cell-free extract under standard assay conditions. Nitrogen source
TABLE 3. Summary of purification of L-amino acid oxidase from Rhodococcus sp. Z-35-1 Activity Protein Specific activity Recovery Purification (unit) (mg) (unit/mg ⋅ protein) (%) (fold) Cell-free extract 36.4 296 0.12 100 1.0 Phenyl-Toyopearl 17.8 35.3 0.50 49 4.1 Phenyl-Toyopearl 16.1 6.66 2.41 44 19.6 DEAE-Toyopearl 14.9 1.16 13.0 41 106 Hydroxyapatite 8.4 0.43 19.1 23 156 Enzyme activity was assayed under standard assay conditions. Specific activity was expressed as units per milligram of protein. Step
units. Substrate specificity and kinetic parameter In the lysine derivatives tested, N α-Z-L-lysine, N α-acetyl-L-lysine and Nε-acetyl-L-lysine were oxidized, but Nα-Z-D-lysine N α-acetyl-D-lysine, and N ε-acetyl-D-lysine were not. Peptides containing L-lysine were oxidized, but those without L-lysine were not. L-Amino acids except for glycine, L-proline and L-cysteine were oxidized, but D-amino acids were not. In these L-amino acids, the reaction rates of L-glutamine and L-asparagine were faster than that of L-glutamic acid and L-aspartic acid, respectively. Amines were also not oxidized (Table 4). These results indicate that this enzyme catalyzed an oxidative deamination of not only ε-amino group of N αacyl-L-lysine but also α-amino group of N ε-acyl-L-lysine. The apparent Km values for N α-Z-L-lysine, N α-acetyl-L-ly-
FIG. 2. Native- and SDS–PAGE of purified enzyme. Lines A, B and C indicate purified enzyme, denatured purified enzyme, and standard proteins, respectively.
sine and N ε-acetyl-L-lysine were estimated to be 12.7 mM, 43.0 mM and 16.3 µM, and their Vmax values were calculated to be 25.8 µmol/min/mg of protein, 6.4 µmol/min/mg of protein and 44.2 µmol/min/mg of protein, respectively. The apparent Km value for L-lysine was estimated to be 62 µM, and its Vmax value was calculated to be 32.0 µmol/min/mg of protein (Table 5). Effects of pH and temperature The effects of pH and temperature on N α-Z-L-lysine oxidation were assayed under standard assay conditions using different pHs and temperatures. The maximum activity was shown at pH 8.0 and at 50°C. Effects of compounds on enzyme activity Effects of compounds on enzyme activity were investigated under standard assay conditions of oxidase activity by adding 1 mM metals, chelating reagents, and other chemicals. Among 10 chemicals, carbonyl reagents such as hydroxylamine, phenylhydrazine and hydrazine greatly inhibited the enzyme activity, but chelating reagents and metals had no significant influence on enzyme activity (Table 6). Cofactor specificity Figure 3 shows the absorption spectrum of purified enzyme solution, which exhibited absorption maxima at around 275, 400 and 460 nm. These results indicate that this enzyme might contain a flavin as a prosthetic group. Then, the purified enzyme solution was heated at 100°C for 3 min to dissociate the flavin coenzyme, and its supernatant was applied to TLC on silica gel with two solvent systems to identify the prosthetic group. The mobility of the flavin component of this oxidase on TLC was same as that of FAD, but not of FMN and riboflavin (Table 7), indicating that this oxidase contained FAD as a prosthetic group, which was not covalently bound to this enzyme. Isoelectric point A single peak of the enzyme activity at pH 4.5 was observed after isoelectric focusing with carrier Ampholine, pH 3.5–10. NH2-terminal amino acid sequence The NH2-terminal sequence of the intact protein was found to be GDLIG
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TABLE 4. Substrate specificity of L-amino acid oxidase from Rhodococcus sp. Z-35-1 Amino acid Relative activity (%) Amino acid 100 L-Phenylalanine N α-Z-L-lysine 10 L-Tyrosine N α-BOC-L-lysine 18 L-Histidine N α-Acetyl-L-lysine 54 L-Tryptophan N ε-Acetyl-L-lysine 0 L-Proline N α-Z-L-asparagine 0 L-Serine N α-Z-L-arginine 0 L-Methionine N α-Z-L-glutamine L-Lysine 53 L-Threonine 58 L-Cystine δ-Hydroxy-L-lysine L-Arginine 61 L-Cysteine L-Asparagine 72 L-Aspartic acid L-Glutamine 73 L-Glutamic acid L-Ornithine 88 Glycine L-Citrulline 64 N α-Z-D-lysine L-Alanine 104 N α-Acetyl-D-lysine L-Leucine 39 N ε-Acetyl-D-lysine L-Isoleucine 15 D-Lysine L-Valine 23 D-Alanine L-2-Aminoadipic acid 46 n-Butylamine Lys-Lys 55 Putrescine Lys-Lys-Lys 14 Hexylamine Lys-Leu 13 Benzylamine Lys-Ala 10 6-Aminohexanoic acid Gly-Lys 13 Gly-Ala Ala-Lys 7 Ala-Gln Enzyme activity was assayed under standard assay conditions using 20 mM amino acids except 2.5 mM L-cystine.
Relative activity (%) 59 87 93 65 0 44 61 4 57 0 3 12 0 0 0 0 <3 0 0 0 0 0 0 0 0
TABLE 5. The Km and Vmax values of L-amino acid oxidase for L-amino acids Vmax (µmol/min/mg of protein) N α-Z-L-lysine 12.7 25.8 43.0 6.4 N α-Acetyl-L-lysine 0.016 44.2 N ε-Acetyl-L-lysine L-Lysine 0.062 32.0 0.456 33.5 δ-Hydroxy-L-lysine L-Ornithine 0.42 27.6 L-Citrulline 0.028 37.2 L-Glutamine 0.25 29.7 L-Glutamic acid 0.97 5.4 Enzyme activity was assayed under standard assay conditions. Amino acid
Km (mM)
KVKGNHSVVILGGGPSGLXXAYELQKAGYKVTVLE (X indicates an unidentified residue). The FAD-binding motif of GXGXXG was confirmed in a position of 17–22 from the NH2-terminus of this enzyme. DISCUSSION We revealed here that conversion of N α-Z-L-lysine into N α-Z-L-AASA in Rhodococcus sp. AIU Z-35-1 was catalyzed by an amino acid oxidase. This enzyme oxidized not only N α-acyl-L-lysine, but also N ε-acyl-L-lysine and L-lysine. The peptidyl L-lysines and L-amino acids except for glycine, L-proline and L-cysteine were also oxidized by this enzyme, whereas N α-acyl-D-lysine, N ε-acyl-D-lysine, D-lysine and D-amino acids were not. Amines were also not oxidized. This enzyme was therefore classified into a group of L-amino acid oxidase (L-amino acid: O2 oxidoreductase
FIG. 3. Absorption spectrum of purified enzyme solution.
[deaminating], EC 1.4.3.2). The L-amino acid oxidases have so far been reported from a variety of sources, and the enzymes from microorganisms had quite strict substrate specificity (4–6). Recently, Brearly et al. (7) have reported an L-amino acid oxidase with rather broad substrate specificity from Bacillus carotarum. Geueke and Hummel (8) have also reported an L-amino acid oxidase with broad substrate specificity from Rhodococcus opacus DSM 43250. When the amino acid sequence similarity was analyzed using 40 amino acid resi-
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FIG. 4. Alignment of amino acid sequence of L-amino acid oxidases and amine oxidases. The NH2-terminal amino acid sequence of L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 was aligned to those of L-amino acid oxidase and to the deduced amino acid sequence of putative L-amino acid oxidase and amine oxidases. Identical amino acid residues are boxed. Rhodococcus sp. Z-35-1 and Rhodococcus opacus DSM 43250 are shown to be L-amino acid oxidase; Rhodococcus sp. RHA 1 is shown to be putative amino acid oxidase; Deinococcus radiodurans R1, Pseudomonas fluorescens pf-5 and Synechococcus sp. JA-2-3′a are shown to be putative mono amine oxidases.
TABLE 6. Effects of chemicals and metals on enzyme activity Chemical Relative activity (%) None 100 Hydroxylamine 45 Hydrazine 13 Phenylhydrazine 0 Semicarbazide 105 o-Phenanthroline 101 EDTA 98 α,α′-Dipyridyl 98 8-Hydroxyquinoline 73 Monoiodoacetic acid 103 N-Ethylmaleimide 102 105 MgCl2 103 NiCl2 108 CoCl2 114 MnCl2 94 FeCl3 Effects of chemicals and metals were assayed under standard assay conditions with 1 mM chemicals or metals. Relative activity was obtained as percent of enzyme activity without chemicals.
TABLE 7. TLC analysis of prosthetic group of L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 Relative mobility Prosthetic group Solvent 1 Solvent 2 Extract of Amino acid oxidase 0.09 0.26 FAD 0.09 0.26 FMN 0.22 0.41 Riboflavin 0.63 0.57 After development at room temperature, spots were detected by ultraviolet irradiation. Solvent 1, n-butanol: acetic acid: water (3: 1:1, by volume); solvent 2, n-butanol :acetone:isopropanol: saturated boric acid (50: 15: 15:30, by volume).
dues from the NH2-terminus of the enzyme from Rhodococcus sp. AIU Z-35-1, more than 80% of the amino acids of this enzyme were identical to that of an L-amino acid oxidase from R. opacus DSM 43250 and a putative L-amino acid oxidase from Rhodococcus sp. RHA 1 (Fig. 4). The enzyme also exhibited similarity to amine oxidases containing FAD as a prosthetic group (more than 50% of the amino acids were identical) (Fig. 4). Previous reports indicated that L-amino acid oxidases were specific to the α-amino group of L-amino acids. However, L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 catalyzed not only the oxidative deamination of the α-amino group of N ε-acyl-L-ly-
sine and L-amino acids but also the ε-amino group of N αacyl-L-lysine, although the apparent Km value for N α-acetylL-lysine was much larger than that for N ε-acetyl-L-lysine. The lysyl oxidases (EC 1.4.3.13) are well known as enzymes catalyzing the oxidative deamination of the ε-amino group of lysine residue in peptides and proteins, but they did not catalyze the oxidative deamination of the α-amino group of L-amino acids. Recently, we have revealed that amine oxidase from Aspergillus niger AKU 3302 catalyzed the oxidative deamination of ε-amino group of N α-Z-L-lysine and N α-Z-D-lysine (9). However, this amine oxidase did not oxidize L-amino acids. Thus, the L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 was different from L-amino acid oxidases, lysyl oxidases and amine oxidases in substrate specificity. Since Rhodococcus sp. AIU Z-35-1 belongs to a group closely related to R. erythropolis but not to R. opacus as shown in Ref. 1, we intend to elucidate the complete amino acid sequence of the L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 in a future study. REFERENCES 1. Isobe, K., Tokuta, K., Narita, Y., Matsuura, A., Sakaguchi, T., and Wakao, N.: Production of N α-benzyloxycarbonylL-aminoadipic acid and N α-benzyloxycarbonyl-D-aminoadipic acid with Rhodococcus sp. AIU Z-35-1. J. Mol. Catal. B, Enzym., 32, 27–32 (2004). 2. Isobe, K., Nagasawa, S., Tokuta, K., Matsuura, A., Sakaguchi, T., and Wakao, N.: A new microbial method for more efficient production of N α-benzyloxycarbonyl-L-aminoadipate δ-semialdehyde and N α-benzyloxycarbonyl-D-aminoadipate δ-semialdehyde. J. Biosci. Bioeng., 100, 288–291 (2005). 3. Laemmli, U. K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680– 685 (1970). 4. Kusakabe, H., Kodama, K., Kuninaka, A., Yoshino, H., Misono, H., and Soda, K.: A novel antitumor enzyme, L-lysine oxidase from Trichoderma viride: purification and enzymological properties. J. Biol. Chem., 255, 976–981 (1980). 5. Koyama, H.: Purification and characterization of a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501. J. Biochem., 92, 1235–1240 (1982). 6. Bohmer, A., Muller, A., Passarge, M., Liebs, P., Honeck, H., and Muller, H-G.: A novel L-glutamate oxidase from Streptomyces endus: purification and properties. Eur. J. Biochem., 182, 327–332 (1989).
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7. Brearly, G. M., Price, C. P., Atkinson, T., and Hammond, P. M.: Purification and partial characterization of a broadrange L-amino acid oxidase from Bacillus carotarum 2Pfa isolated from soil. Appl. Microbiol. Biotechnol., 41, 670–676 (1994). 8. Geueke, B. and Hunmmel, W.: A new bacterial L-amino acid oxidase with a broad substrate specificity: purification and characterization. Enzyme Microb. Technol., 31, 77–87 (2002). 9. Isobe, K., Tokuta, K., Narita, Y., Matsuura, A., Sakaguchi, T., and Wakao, N.: A method for production of N α-benzyloxycarbonyl-aminoadipate-δ-semialdehyde with amine oxidase from Aspergillus niger. J. Mol. Catal. B, Enzym., 30, 119–123 (2004). 10. McLeod, M. P., Warren, R. L., Hsiao, W. W. L., Araki, N., Myhre, M., Fernandes, C., Miyazawa, D., Wong, W., Lillquist, A. L., Wang, D., and other 19 authors: The complete genome of Rhodococcus sp. RHA1 provides insights
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into a catabolic powerhouse. Proc. Natl. Acad. Sci. USA, 103, 15582–15587 (2006). 11. White, O., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., Nelson, W. C., Richardson, D. L., and other 22 authors: Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science, 286, 1571–1577 (1999). 12. Paulsen, I. T., Press, C. M., Ravel, J., Kobayashi, D. Y., Myers, G. S. A., Mavrodi, D. V., DeBoy, R. T., Seshadri, R., Ren, Q., Madupu, R., and other 19 authors: Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol., 23, 873–878 (2005). 13. Allewalt, J. P., Bateson, M. M., Revsbech, N. P., Slack, K., and Ward, D. M.: Effect of temperature and light on growth of and photosynthesis by Synechococcus isolates typical of those predominating in the octopus spring microbial mat community of Yellowstone National Park. Appl. Environ. Microbiol., 72, 544–550 (2006).
© 2007, The Society for Biotechnology, Japan
Characterization of N α-Benzyloxycarbonyl-L-Lysine Oxidizing Enzyme from Rhodococcus sp. AIU Z-35-1 Kimiyasu Isobe1* and Shouko Nagasawa1 Department of Agro-bioscience, Faculty of Agriculture, Iwate University, 3 Ueda, Morioka 020-8550, Japan1 Received 19 April 2007/Accepted 23 June 2007
An oxidase catalyzing conversion of N α-benzyloxycarbonyl-L-lysine (N α-Z-L-lysine) to N α-benzyloxycarbonyl-L-aminoadipate-δ-semialdehyde (N α-Z-L-AASA) was purified from Rhodococcus sp. AIU Z-35-1, and its properties were revealed. This enzyme catalyzed an oxidative deamination of the ε-amino group of N α-acyl-L-lysine and the α-amino group of N ε-acyl-L-lysine. The apparent Km value for N α-acetyl-L-lysine was much larger than that for N ε-acetyl-L-lysine. The peptidyl L-lysines, L-lysine and many other L-amino acids were also oxidized, but N α-acyl-D-lysine, N ε-acylD-lysine and D-amino acids were not. Thus, the conversion of N α-Z-L-lysine into N α-Z-L-AASA was catalyzed by the L-amino acid oxidase with broad substrate specificity. This enzyme, a flavoprotein with a molecular mass of 100 kDa, consisted of two identical subunits of 51 kDa. [Key words: N α-benzyloxycarbonyl-L-lysine, N α-benzyloxycarbonyl-L-aminoadipate-δ-semialdehyde, N α-benzyloxycarbonyl-L-aminoadipic acid, L-amino acid oxidase]
Recently, we isolated a new bacterial strain, named Rhodococcus sp. AIU Z-35-1, for production of N α-benzyloxycarbonyl-L-aminoadipic acid (N α-Z-L-AAA), and a new biochemical method for N α-Z-L-AAA production has been developed using this isolated strain (1). Since this strain converted N α-benzyloxycarbonyl-L-lysine (N α-Z-L-lysine) to N α-Z-L-AAA via N α-benzyloxycarbonyl-L-aminoadipateδ-semialdehyde (N α-Z-L-AASA), it was also useful for the production of N α-Z-L-AASA (2). Although these new bacterial methods were superior to chemical and other biochemical methods in the conversion yields and formation speed of N α-Z-L-AASA and N α-Z-L-AAA, the enzymes catalyzing the conversion of N α-Z-L-lysine to N α-Z-L-AAA have not been identified. We therefore investigated the enzymes catalyzing this conversion, and found an enzyme exhibiting oxidase activity on N α-Z-L-lysine. The present paper describes production, purification and some remarkable properties of the N α-Z-L-lysine oxidizing enzyme produced by Rhodococcus sp. AIU Z-35-1.
Sanyo Fine (Osaka). Peroxidase was gift from Amano Enzyme (Nagoya). All other chemicals used were of analytical grade and commercially available. Cultivation of strain Z-35-1 Rhodococcus sp. Z-35-1 was first incubated in test tube containing 5 ml of N α-Z-L-lysine medium, pH 7.0, consisting of 0.2% KH2PO4, 0.1% Na2HPO4, 0.05% MgSO4 ⋅ 7H2O, 0.5% glucose, and 0.5% N α-Z-L-lysine at 30°C for 2 d with shaking (120 strokes/min). The culture (1.5 ml) was then inoculated into 500 ml shaker flask containing 150 ml of the N α-ZL-lysine medium, and it was cultivated at 30°C for 2 d with shaking. This strain was also cultivated in the L-lysine medium, which consisted of the same as N α-Z-L-lysine medium except that N α-ZL-lysine was replaced by L-lysine, under the same conditions as the N α-Z-L-lysine medium. Then, 20 ml of the second culture was transferred into a 3-l of culture flask containing 2 l of the L-lysine medium. After the third culture was carried out at 30°C for 1 d, the cells were harvested by centrifugation at 20,000 ×g for 10 min, washed with 50 mM potassium phosphate buffer, pH 7.0, and then stored at −20°C until use. Analysis of oxidase activity Oxidase activity was assayed by measuring the formation rate of hydrogen peroxide as follows. The standard reaction mixture contained 40 µmol of N α-Z-L-lysine, 0.6 µmol of 4-aminoantipyrine, 1.94 µmol of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt dihydrate, 6.7 units of peroxidase, 0.1 mmol of potassium phosphate, pH 7.0, and an appropriate amount of enzyme, in a final volume of 1.0 ml. The formation of hydrogen peroxide was spectrophotometrically followed at 30°C for 5 min by measuring the absorbance at 555 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one micromole of hydrogen peroxide per min. Assay of dehydrogenase activity Dehydrogenase activity was assayed by measuring the formation rate of NAD(P)H as follows. The standard reaction mixture contained 40 µmol of Nα-ZL-lysine, 0.6 µmol of β-NAD+ or β-NADP+, 0.37 µmol of nitro
MATERIALS AND METHODS Chemicals L-Lysine and other L-amino acids were purchased from Wako Pure Chemicals (Osaka). N α-Z-L-lysine and N α-Z-D-lysine were purchased from Calbiochem-Novabiochem (Läufelfingen, Switzerland) and Fluka Chemie (Buchs, Switzerland), respectively. Peptidyl L-lysines were obtained from Sigma Chemical Japan (Tokyo). N α-Acetyl-L-lysine, N ε-acetyl-L-lysine and N α-acetyl-D-lysine were from Watanabe Chemical Industries (Hiroshima). N α-ZL-glutamine, N α-Z-L-arginine and N α-Z-L-asparagine were from * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)19-621-6155 218
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blue tetrazolium, 0.2 unit of diaphorase, 0.1 mmol of potassium phosphate, pH 7.0, 0.05% Triton X-100 and an appropriate amount of enzyme, in a final volume of 1.0 ml. The formation of NAD(P)H was spectrophotometrically followed at 30°C by measuring the absorbance at 550 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one micromole of NAD(P)H per min. Purification of enzyme Purification of enzyme was carried out using cells obtained from 24 l culture broth of the L-lysine medium. All procedures were done at 5–10°C. The buffer used was potassium phosphate buffer, pH 7.0. Preparation of cell-free extract Cells (wet weight, 14.7 g) were suspended in 250 ml of 10 mM buffer, and disrupted by a Multi-beads shocker (Yasui Kikai, Osaka) at 2500 rpm for 8 min (2 min × 4). The supernatant (250 ml) was obtained by centrifugation at 20,000 ×g for 10 min. Ammonium sulfate fractionation Solid ammonium sulfate was added to 50% saturation in the supernatant, and resulting precipitate was discarded by centrifugation at 20,000 ×g for 10 min. First Phenyl-Toyopearl column chromatography (stepwise elution) A Phenyl-Toyopearl resin equilibrated with 10 mM buffer containing 1.5 M ammonium sulfate was added into the supernatant, and its suspension was stirred for 1 h. Using this suspension, the column (16 × 2.2 cm diameter) was prepared, and undesirable proteins were washed out with 10 mM buffer containing 1.5 M ammonium sulfate. The oxidase was then eluted with 10 mM buffer containing 0.8 M ammonium sulfate. The active fractions were collected, and ammonium sulfate was added to1.5 M. Second Phenyl-Toyopearl column chromatography The eluate from the first Phenyl-Toyopearl column chromatography was applied again to a Phenyl-Toyopearl column (16 ×2.2 cm diameter) equilibrated with 10 mM buffer containing 1.5 M ammonium sulfate. After the column was washed with the same buffer, the adsorbed enzyme was eluted by a linear gradient with 10 mM buffer containing 1.5 M ammonium sulfate and 0.8 M ammonium sulfate (300 ml each), followed with 150 ml of 10 mM buffer containing 0.8 M ammonium sulfate. The active fractions were collected, and deionized to the conductivity of 1.8 ms/cm by ultrafiltration. DEAE-Toyopearl column chromatography The deionized enzyme solution was applied to a DEAE-Toyopearl column (16 ×1.8 cm diameter) equilibrated with 10 mM buffer, and the column was washed with 10 mM buffer. The adsorbed enzyme was then eluted by a linear gradient with 10 mM buffer and 0.15 M NaCl (200 ml each). The active fractions were collected, and dialyzed against 5 mM buffer. Hydroxyapatite column chromatography The dialyzed enzyme solution was applied to a Hydroxyapatite column (13 ×1.0 cm diameter) equilibrated with 10 mM buffer, and the column was washed with the same buffer. The adsorbed enzyme was then eluted by a linear gradient with 10 mM buffer and 100 mM buffer (50 ml each), followed with 20 ml of 100 mM buffer. The active fractions were collected, and the purity was analyzed. Protein measurement Protein concentration was measured with a Protein Quantification Kit (Dojindo Laboratories, Tokyo). SDS–PAGE and molecular mass SDS–PAGE was performed according to the method of Laemmli (3). Proteins were stained with Coomassie Brilliant Blue R-250. Molecular mass was estimated by gel filtration on a TSK gel G3000SWXL column and by SDS–PAGE using standard markers of molecular mass (Sigma Japan). Isoelectric point The isoelectric point was determined with an isoelectric focusing apparatus (Nippon Eido, Tokyo) under conditions of 1% Ampholine, pH 3.5–10, with a sucrose gradient at 400 V for 2 d at 4°C. One-milliliter fractions were collected, and the pH was measured at 4°C. NH2-terminal amino acid sequence The amino acid sequence
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of the intact enzyme was determined using Applied Biosystems gas-phase protein sequencer equipped with an on-line reversephase chromatography system for identification of PTH-amino acids.
RESULTS Identification of enzyme Enzyme catalyzing conversion of N α-Z-L-lysine into N α-Z-L-AASA was analyzed using cell-free extract, which was prepared from cells incubated for 36 h in the N α-Z-L-lysine medium. The oxidase activity on N α-Z-L-lysine was obtained, but the dehydrogenase activity on N α-Z-L-lysine was less than 2% of the oxidase activity (Table 1). In addition, formation of equimolecular amounts of NH3 and H2O2 was confirmed (data not shown). It was therefore concluded that the conversion of N α-Z-L-lysine into N α-Z-L-AASA was catalyzed by an oxidase according to Fig. 1. Effects of nitrogen source on enzyme production Rhodococcus sp. AIU Z-35-1 was incubated at 30°C for 36 h in the medium containing N α-Z-L-lysine, L-lysine, n-butylamine, ammonium nitrate or ammonium sulfate as a nitrogen source. This strain grew well in the medium containing Nα-Z-L-lysine, L-lysine, ammonium nitrate or ammonium sulfate, and the N α-Z-L-lysine oxidase activity was obtained by culturing with N α-Z-L-lysine or L-lysine, but not with the other compounds tested (Table 2). In addition, the oxidase activity obtained from the L-lysine medium was four times higher than that of the Nα-Z-L-lysine medium. The L-lysine medium was therefore suitable for enzyme production. Purification and molecular mass The purification procedure is summarized in Table 3. The oxidase was purified to an electrophoretically homogeneous state by approximately 155-fold. The purified enzyme showed a single protein band on native- and SDS–PAGE (Fig. 2). The molecular mass of the native and denatured enzymes was estimated to be 100 kDa on TSK gel G3000SWXL column and 51 kDa on SDS–PAGE, respectively (data not shown). These results indicate that this enzyme consisted of two identical subTABLE 1. Identification of enzyme catalyzing conversion of N α-Z-L-lysine to N α-Z-L-AASA Enzyme activity (m unit/100 ml broth) Dehydrogenase Oxidase NADP+ NAD+ α N -Z-L-lysine 13.0 0.21 0 Rhodococcus sp. AIU Z-35-1 was cultured at 30°C for 36 h in the N α-Z-L-lysine medium. Cells of 100 ml culture broth were disrupted with Multi-beads shocker at 2500 rpm for 4 min, and cell debris was discarded by centrifugation at 20,000 × g for 10 min. Enzyme activity was assayed using supernatant under standard assay conditions. Substrate
FIG. 1. Conversion of N α-Z-L-lysine into N α-Z-L-AASA by L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1.
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TABLE 2. Effects of nitrogen source on enzyme production Oxidase activity Protein Specific activity (m unit/100 ml ⋅broth) (mg/100 ml ⋅broth) (m unit/mg ⋅ protein) 15 15.8 0.95 N α-Z-L-lysine L-Lysine 60 21.4 2.80 n-Butylamine 1> 0.27 0 Ammonium sulfate 1> 58.2 0 Ammonium nitrate 1> 17.2 0 Rhodococcus sp. AIU Z-35-1 was incubated at 30°C for 36 h in the medium containing indicated chemicals as a nitrogen source. N α-Z-L-lysine oxidase activity was assayed using cell-free extract under standard assay conditions. Nitrogen source
TABLE 3. Summary of purification of L-amino acid oxidase from Rhodococcus sp. Z-35-1 Activity Protein Specific activity Recovery Purification (unit) (mg) (unit/mg ⋅ protein) (%) (fold) Cell-free extract 36.4 296 0.12 100 1.0 Phenyl-Toyopearl 17.8 35.3 0.50 49 4.1 Phenyl-Toyopearl 16.1 6.66 2.41 44 19.6 DEAE-Toyopearl 14.9 1.16 13.0 41 106 Hydroxyapatite 8.4 0.43 19.1 23 156 Enzyme activity was assayed under standard assay conditions. Specific activity was expressed as units per milligram of protein. Step
units. Substrate specificity and kinetic parameter In the lysine derivatives tested, N α-Z-L-lysine, N α-acetyl-L-lysine and Nε-acetyl-L-lysine were oxidized, but Nα-Z-D-lysine N α-acetyl-D-lysine, and N ε-acetyl-D-lysine were not. Peptides containing L-lysine were oxidized, but those without L-lysine were not. L-Amino acids except for glycine, L-proline and L-cysteine were oxidized, but D-amino acids were not. In these L-amino acids, the reaction rates of L-glutamine and L-asparagine were faster than that of L-glutamic acid and L-aspartic acid, respectively. Amines were also not oxidized (Table 4). These results indicate that this enzyme catalyzed an oxidative deamination of not only ε-amino group of N αacyl-L-lysine but also α-amino group of N ε-acyl-L-lysine. The apparent Km values for N α-Z-L-lysine, N α-acetyl-L-ly-
FIG. 2. Native- and SDS–PAGE of purified enzyme. Lines A, B and C indicate purified enzyme, denatured purified enzyme, and standard proteins, respectively.
sine and N ε-acetyl-L-lysine were estimated to be 12.7 mM, 43.0 mM and 16.3 µM, and their Vmax values were calculated to be 25.8 µmol/min/mg of protein, 6.4 µmol/min/mg of protein and 44.2 µmol/min/mg of protein, respectively. The apparent Km value for L-lysine was estimated to be 62 µM, and its Vmax value was calculated to be 32.0 µmol/min/mg of protein (Table 5). Effects of pH and temperature The effects of pH and temperature on N α-Z-L-lysine oxidation were assayed under standard assay conditions using different pHs and temperatures. The maximum activity was shown at pH 8.0 and at 50°C. Effects of compounds on enzyme activity Effects of compounds on enzyme activity were investigated under standard assay conditions of oxidase activity by adding 1 mM metals, chelating reagents, and other chemicals. Among 10 chemicals, carbonyl reagents such as hydroxylamine, phenylhydrazine and hydrazine greatly inhibited the enzyme activity, but chelating reagents and metals had no significant influence on enzyme activity (Table 6). Cofactor specificity Figure 3 shows the absorption spectrum of purified enzyme solution, which exhibited absorption maxima at around 275, 400 and 460 nm. These results indicate that this enzyme might contain a flavin as a prosthetic group. Then, the purified enzyme solution was heated at 100°C for 3 min to dissociate the flavin coenzyme, and its supernatant was applied to TLC on silica gel with two solvent systems to identify the prosthetic group. The mobility of the flavin component of this oxidase on TLC was same as that of FAD, but not of FMN and riboflavin (Table 7), indicating that this oxidase contained FAD as a prosthetic group, which was not covalently bound to this enzyme. Isoelectric point A single peak of the enzyme activity at pH 4.5 was observed after isoelectric focusing with carrier Ampholine, pH 3.5–10. NH2-terminal amino acid sequence The NH2-terminal sequence of the intact protein was found to be GDLIG
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TABLE 4. Substrate specificity of L-amino acid oxidase from Rhodococcus sp. Z-35-1 Amino acid Relative activity (%) Amino acid 100 L-Phenylalanine N α-Z-L-lysine 10 L-Tyrosine N α-BOC-L-lysine 18 L-Histidine N α-Acetyl-L-lysine 54 L-Tryptophan N ε-Acetyl-L-lysine 0 L-Proline N α-Z-L-asparagine 0 L-Serine N α-Z-L-arginine 0 L-Methionine N α-Z-L-glutamine L-Lysine 53 L-Threonine 58 L-Cystine δ-Hydroxy-L-lysine L-Arginine 61 L-Cysteine L-Asparagine 72 L-Aspartic acid L-Glutamine 73 L-Glutamic acid L-Ornithine 88 Glycine L-Citrulline 64 N α-Z-D-lysine L-Alanine 104 N α-Acetyl-D-lysine L-Leucine 39 N ε-Acetyl-D-lysine L-Isoleucine 15 D-Lysine L-Valine 23 D-Alanine L-2-Aminoadipic acid 46 n-Butylamine Lys-Lys 55 Putrescine Lys-Lys-Lys 14 Hexylamine Lys-Leu 13 Benzylamine Lys-Ala 10 6-Aminohexanoic acid Gly-Lys 13 Gly-Ala Ala-Lys 7 Ala-Gln Enzyme activity was assayed under standard assay conditions using 20 mM amino acids except 2.5 mM L-cystine.
Relative activity (%) 59 87 93 65 0 44 61 4 57 0 3 12 0 0 0 0 <3 0 0 0 0 0 0 0 0
TABLE 5. The Km and Vmax values of L-amino acid oxidase for L-amino acids Vmax (µmol/min/mg of protein) N α-Z-L-lysine 12.7 25.8 43.0 6.4 N α-Acetyl-L-lysine 0.016 44.2 N ε-Acetyl-L-lysine L-Lysine 0.062 32.0 0.456 33.5 δ-Hydroxy-L-lysine L-Ornithine 0.42 27.6 L-Citrulline 0.028 37.2 L-Glutamine 0.25 29.7 L-Glutamic acid 0.97 5.4 Enzyme activity was assayed under standard assay conditions. Amino acid
Km (mM)
KVKGNHSVVILGGGPSGLXXAYELQKAGYKVTVLE (X indicates an unidentified residue). The FAD-binding motif of GXGXXG was confirmed in a position of 17–22 from the NH2-terminus of this enzyme. DISCUSSION We revealed here that conversion of N α-Z-L-lysine into N α-Z-L-AASA in Rhodococcus sp. AIU Z-35-1 was catalyzed by an amino acid oxidase. This enzyme oxidized not only N α-acyl-L-lysine, but also N ε-acyl-L-lysine and L-lysine. The peptidyl L-lysines and L-amino acids except for glycine, L-proline and L-cysteine were also oxidized by this enzyme, whereas N α-acyl-D-lysine, N ε-acyl-D-lysine, D-lysine and D-amino acids were not. Amines were also not oxidized. This enzyme was therefore classified into a group of L-amino acid oxidase (L-amino acid: O2 oxidoreductase
FIG. 3. Absorption spectrum of purified enzyme solution.
[deaminating], EC 1.4.3.2). The L-amino acid oxidases have so far been reported from a variety of sources, and the enzymes from microorganisms had quite strict substrate specificity (4–6). Recently, Brearly et al. (7) have reported an L-amino acid oxidase with rather broad substrate specificity from Bacillus carotarum. Geueke and Hummel (8) have also reported an L-amino acid oxidase with broad substrate specificity from Rhodococcus opacus DSM 43250. When the amino acid sequence similarity was analyzed using 40 amino acid resi-
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FIG. 4. Alignment of amino acid sequence of L-amino acid oxidases and amine oxidases. The NH2-terminal amino acid sequence of L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 was aligned to those of L-amino acid oxidase and to the deduced amino acid sequence of putative L-amino acid oxidase and amine oxidases. Identical amino acid residues are boxed. Rhodococcus sp. Z-35-1 and Rhodococcus opacus DSM 43250 are shown to be L-amino acid oxidase; Rhodococcus sp. RHA 1 is shown to be putative amino acid oxidase; Deinococcus radiodurans R1, Pseudomonas fluorescens pf-5 and Synechococcus sp. JA-2-3′a are shown to be putative mono amine oxidases.
TABLE 6. Effects of chemicals and metals on enzyme activity Chemical Relative activity (%) None 100 Hydroxylamine 45 Hydrazine 13 Phenylhydrazine 0 Semicarbazide 105 o-Phenanthroline 101 EDTA 98 α,α′-Dipyridyl 98 8-Hydroxyquinoline 73 Monoiodoacetic acid 103 N-Ethylmaleimide 102 105 MgCl2 103 NiCl2 108 CoCl2 114 MnCl2 94 FeCl3 Effects of chemicals and metals were assayed under standard assay conditions with 1 mM chemicals or metals. Relative activity was obtained as percent of enzyme activity without chemicals.
TABLE 7. TLC analysis of prosthetic group of L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 Relative mobility Prosthetic group Solvent 1 Solvent 2 Extract of Amino acid oxidase 0.09 0.26 FAD 0.09 0.26 FMN 0.22 0.41 Riboflavin 0.63 0.57 After development at room temperature, spots were detected by ultraviolet irradiation. Solvent 1, n-butanol: acetic acid: water (3: 1:1, by volume); solvent 2, n-butanol :acetone:isopropanol: saturated boric acid (50: 15: 15:30, by volume).
dues from the NH2-terminus of the enzyme from Rhodococcus sp. AIU Z-35-1, more than 80% of the amino acids of this enzyme were identical to that of an L-amino acid oxidase from R. opacus DSM 43250 and a putative L-amino acid oxidase from Rhodococcus sp. RHA 1 (Fig. 4). The enzyme also exhibited similarity to amine oxidases containing FAD as a prosthetic group (more than 50% of the amino acids were identical) (Fig. 4). Previous reports indicated that L-amino acid oxidases were specific to the α-amino group of L-amino acids. However, L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 catalyzed not only the oxidative deamination of the α-amino group of N ε-acyl-L-ly-
sine and L-amino acids but also the ε-amino group of N αacyl-L-lysine, although the apparent Km value for N α-acetylL-lysine was much larger than that for N ε-acetyl-L-lysine. The lysyl oxidases (EC 1.4.3.13) are well known as enzymes catalyzing the oxidative deamination of the ε-amino group of lysine residue in peptides and proteins, but they did not catalyze the oxidative deamination of the α-amino group of L-amino acids. Recently, we have revealed that amine oxidase from Aspergillus niger AKU 3302 catalyzed the oxidative deamination of ε-amino group of N α-Z-L-lysine and N α-Z-D-lysine (9). However, this amine oxidase did not oxidize L-amino acids. Thus, the L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 was different from L-amino acid oxidases, lysyl oxidases and amine oxidases in substrate specificity. Since Rhodococcus sp. AIU Z-35-1 belongs to a group closely related to R. erythropolis but not to R. opacus as shown in Ref. 1, we intend to elucidate the complete amino acid sequence of the L-amino acid oxidase from Rhodococcus sp. AIU Z-35-1 in a future study. REFERENCES 1. Isobe, K., Tokuta, K., Narita, Y., Matsuura, A., Sakaguchi, T., and Wakao, N.: Production of N α-benzyloxycarbonylL-aminoadipic acid and N α-benzyloxycarbonyl-D-aminoadipic acid with Rhodococcus sp. AIU Z-35-1. J. Mol. Catal. B, Enzym., 32, 27–32 (2004). 2. Isobe, K., Nagasawa, S., Tokuta, K., Matsuura, A., Sakaguchi, T., and Wakao, N.: A new microbial method for more efficient production of N α-benzyloxycarbonyl-L-aminoadipate δ-semialdehyde and N α-benzyloxycarbonyl-D-aminoadipate δ-semialdehyde. J. Biosci. Bioeng., 100, 288–291 (2005). 3. Laemmli, U. K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680– 685 (1970). 4. Kusakabe, H., Kodama, K., Kuninaka, A., Yoshino, H., Misono, H., and Soda, K.: A novel antitumor enzyme, L-lysine oxidase from Trichoderma viride: purification and enzymological properties. J. Biol. Chem., 255, 976–981 (1980). 5. Koyama, H.: Purification and characterization of a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501. J. Biochem., 92, 1235–1240 (1982). 6. Bohmer, A., Muller, A., Passarge, M., Liebs, P., Honeck, H., and Muller, H-G.: A novel L-glutamate oxidase from Streptomyces endus: purification and properties. Eur. J. Biochem., 182, 327–332 (1989).
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