Enzymatic Characterization of an Amine Oxidase from Arthrobacter Sp

80365 (381)

Biosci. Biotechnol. Biochem., 72, 80365-1–7, 2008

Enzymatic Characterization of an Amine Oxidase from Arthrobacter sp. Used to Measure Phosphatidylethanolamine

Hiroko OTA,1;* Hideto TAMEZANE,2;* Yoshie SASANO,2 Eisaku HOKAZONO,2 y Yuko YASUDA,1 Shin-ichi SAKASEGAWA,1; Shigeyuki IMAMURA,1 Tomohiro TAMURA,3;4 and Susumu OSAWA2

1Asahi Kasei Pharma Corporation, Shizuoka 410-4321, Japan 2Kyushu University, Division of Biological Science and Technology, Department of Health Science, School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 3Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Sciences and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan 4Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan

Received May 29, 2008; Accepted July 1, 2008; Online Publication, October 7, 2008 [doi:10.1271/bbb.80365]Advance View

Ethanolamine oxidase was screened with the aim of most abundant membrane phospholipid in mammals, using it to establish a novel enzymatic phosphatidyle- accounting for 20% to 50% of total phospholipids. thanolamine assay. Ethanolamine oxidase activity was Together with phosphatidylserine, the most abundant detected in the crude extract of Arthrobacter sp., and the phospholipid in mammalian membranes, PE plays key enzyme was purified more than 15-fold in three steps roles in a variety of biological processes, including with a 54% yield. SDS–PAGE revealed the presence of apoptosis and cell signaling.1) The conventional methods only one band, which migrated, with an apparent for assaying PE are thin-layer chromatography2) and Proofs3) molecular mass of 70 kDa. Biochemical characterization HPLC-based analysis, but, these methods are too time of the enzyme showed phenylethylamine to be the consuming to be used in routine work. In the present preferred substrate, with the highest kcat/Km value. study, therefore, our aim was to begin to develop a The primary structure, determined by sequencing the convenient new PE assay that can be used routinely to cloned gene, showed a high degree of identity to Cu- analyze PE levels in, for example, serum and plasma. containing phenylethylamine oxidase (64%). When Our strategy for this assay was to employ a contin- heterologously overexpressed in Escherichia coli, the uously coupled reaction consisting of three independent enzyme exhibited only a trace of amine oxidase activity, enzymatic reactions: (i) hydrolysis of the phosphodiester but high levels of activity emerged after exposure to bond of PE using phospholipase D (EC 3.1.4.4) Cu2þ, as is typical of recombinant copper amine to generate phosphatidic acid and ethanolamine,4) (ii) oxidases. Preliminary application of this enzyme cou- oxidative deamination of the ethanolamine with con- pled with phospholipase D for determination of phos- comitant reduction of O2 to generate glycolaldehyde, phatidylethanolamine is also described. This is the first NH3, and H2O2, and (iii) an oxidative coupling reaction enzymatic method for the measurement of phosphati- with 4-aminoantipyrine and, perhaps, phenol catalyzed dylethanolamine. by peroxidase (EC 1.11.1.7) in the presence of H2O2. In this scheme, an enzyme that can oxidize ethanol- Key words: copper amine oxidase; Arthrobacter sp.; amine is required, and the object of the present study phosphatidylethanolamine was to express and purify an ethanolamine oxidative enzyme. Phosphatidylethanolamine (PE) is an aminophospho- Two classes of phylogenetically unrelated enzymes lipid found in biological membranes. It is the second are known to catalyze the oxidation of monoamines with

* These authors contributed equally to this work. y To whom correspondence should be addressed. Fax: +81-558-76-7149; E-mail: [email protected] Abbreviations: PE, phosphatidylethanolamine; TPQ, 2,4,5-trihydroxyphenylalanine quinone; AAO, Arthrobacter sp. copper amine oxidase; LB, Luria-Bertani; TOOS, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine; AGAO, Arthrobacter globiformis phenylethylamine oxidase 80365-2 H. OTA et al. broad substrate specificity. These enzymes are flavin After the cell debris was removed by centrifugation containing amine oxidoreductase (Pfam PF01593, EC (15;000 g for 60 min), the supernatant was collected as 1.4.3.4), and copper amine oxidase (PF01179, EC the crude extract. Thereafter, solid (NH4)2SO4 was 1.4.3.6). The reactions catalyzed by these two enzymes added to a concentration of 18% (w/v), and the resulting are formally identical, and the products are correspond- mixture was centrifuged at 10;000 g for 15 min to pellet- ing aldehydes, NH3, and H2O2. Copper amine oxidases out protein. Again the supernatant was collected, and are dimers comprised of identical 70–90 kDa subunits, this time solid (NH4)2SO4 was added, to a concentration each of which contains a single copper ion and a of 12%, after which the mixture was centrifuged at covalently bound cofactor formed through post-transla- 10;000 g for 15 min to precipitate the AAO. The tional modification of a tyrosine side chain to 2,4,5- precipitated AAO was dissolved in the standard buffer trihydroxyphenylalanine quinone (TPQ).5,6) Copper and dialyzed against 50 volumes of the same buffer. The amine oxidase has been purified from a wide variety dialyzate, containing the enzyme, was loaded onto 2 of organisms and characterized,7) and the crystal struc- liters of DEAE-Sepharose Fast Flow gel packed in a tures of the enzymes from Arthrobacter globiformis8) BPG100 column (10 cm column i.d., GE Healthcare, and Escherichia coli9) have been solved. Tokyo) equilibrated with the buffer. After the column We detected ethanolamine oxidase activity in a cell- was washed with the same buffer, the enzyme was free crude extract of Arthrobacter sp., and the purified eluted with a linear gradient of 0–0.5 M NaCl. The enzyme was found to be a copper amine oxidase (AAO). fractions containing the enzyme were dialyzed against Here we describe this enzyme’s biochemical character- 20 volumes of the standard buffer, and the enzyme ization and its heterologous overexpression in E. coli.In solution was loaded onto 0.5 liters of Q-Sepharose High addition, preliminary application of this enzyme coupled Performance gel packed in a XK50 column (5 cm with phospholipase D for determination of PE is also column i.d., GE Healthcare) equilibrated with the same described.Advance Viewbuffer. After the column was washed with the buffer, the bound enzyme was eluted with a linear gradient of Materials and Methods 0–0.5 M KCl. The resulting enzyme solution was con- centrated using a 30-kDa centrifugal filter device Microorganism and growth conditions. Arthrobacter (Millipore, Bedford, MA), and dialyzed against 100 sp. (FERM P-06240, BP-0421), originally isolated from volumes of buffer. soil, was used as the source of AAO and its chromo- somal DNA. The cells were grown in a medium Analyses of N-terminal and inner amino acid se- containing 1.0% yeast extract, 1.0% meat extract, quences. Once purified, the enzyme was denatured using 1.0% peptone, 1.0% corn steep liquor, and 0.05% 1 M urea and then digested with Lys-C or Asp-N Proofs phenylethylamine at pH 7.5. The bacterium was aerobi- endopeptidase (Roche) for 17 h at 37 C. The digested cally cultivated for 40 h at 30 C. fragments were then purified on a reverse-phase Capcell Pak C18 HPLC column (Shiseido, Tokyo) using a Enzyme assay and protein measurement. Unless solvent system composed of a 50-min linear gradient of otherwise specified, the standard reaction mixture con- 10% to 70% acetonitrile containing 0.1% trifluoroacetic tained 20 mM potassium phosphate (pH 7.5), 0.1 mM acid. The flow rate was 0.5 ml/min, and the eluate was tyramine, 0.03% 4-aminoantipyrine, 0.02% N,N-Bis(4- monitored at an absorbance of 215 nm. The overall sulfobutyl)-3-methylaniline (Dojin, Kumamoto, Japan), amino acid sequences of these peptide fragments and the and 5 U/ml peroxidase (Sigma, St. Louis, MO) in a total N-terminal region of the purified AAO were analyzed volume of 1.0 ml. The reaction was started by the by automated Edman degradation using an Applied addition of 15 ml of enzyme solution. The reaction Biosystems gas-phase protein sequencer. mixture was incubated at 37 C in a cuvette, and the increase in absorbance at 546 nm was followed spec- Cloning and sequencing of the AAO gene. A 500-bp 1 trophotometrically (d ¼ 1 cm and "546 ¼ 16 mM fragment of the AAO gene was amplified using cm1). One U of enzyme was defined as the amount oligonucleotide primers corresponding to the double- catalyzing the oxidation of 1 mmole of tyramine per min underlined inner peptides shown in Fig. 2 (sense, 50-ggn at pH 7.5 at 37 C. Protein was measured using a Bio- gcn ccn tty cay car cay ytn tty gg-30, and antisense, 50- Rad protein assay kit (Bio-Rad, Hercules, CA); bovine acn cay tty ccn mgn acn gar gay gay cc-30). Chromo- serum albumin served as the standard protein. somal DNA isolated from Arthrobacter sp. as described previously10) served as the template. For genome Purification of amine oxidase from Arthrobacter sp. walking to clone the missing part of the AAO gene, an All operations were carried out at room temperature, and LA PCR in vitro Cloning Kit (Takara, Shiga, Japan) was 10 mM potassium phosphate buffer (pH 7.0) was used as used as instructed by the manufacturer.11) The entire the standard buffer throughout purification. Arthrobacter AAO gene was then amplified using oligonucleotide sp. cells were lysed in buffer containing 0.5% lysozyme, primers 50-ttt aca tgt ctg aaa cca ccg ccg tca ata-30 1mM EDTA, and 0.05% Triton X-100 for 1 h at 38 C. (sense) and 50-ttt agt act cta gcc gtt aga acc gca gca gcc Amine Oxidase from Arthrobacter sp. 80365-3 a-30 (antisense), which contain a unique Afl III and a diester bond of PE, oxidative deamination of the unique Sca I restriction site respectively (underlined). ethanolamine, and the oxidative coupling reaction with The amplified 2-kb fragment was digested with Afl III 4-aminoantipyrine and TOOS; it contained 100 mM and Sca I and ligated with pTV118N vector (Takara) Tris–HCl pH 7.5, 2 U/ml phospholipase D (Asahi Kasei 4) linearized with Nco I and Sca I to generate pTV118N/ Pharma, Tokyo), 1.5 mM CaCl2, 0.78 mM 4-amino- AAO. E. coli TOP 10 cells (Invitrogen, La Jolla, CA) antipyrine, and 0.3% Triton X-100. The buffer systems were then transformed with the pTV118N/AAO and of the reaction mixtures were selected for pH optima and spread onto an LB agar plate containing 50 mg/l of the pH stability of AAO, peroxidase, and phospholipase ampicillin. Positive colonies were selected by the colony D.4) The automated assays were performed using a PCR method, and then cultured in liquid LB medium Hitachi 7170 analyzer (Hitachi, Tokyo). In each assay, containing 50 mg/l of ampicillin. Plasmid pTV118N/ 200 ml of mixture #1 was incubated with 30 mlof AAO containing the AAO gene was then isolated and samples for 5 min at 37 C, after which 100 ml of mixture used to transform E. coli strains W3110, LE392, JM101, #2 was added. After the addition of mixture #2, the JM109, and TH2 (Toyobo, Osaka, Japan). increasing absorbance at 546 nm was measured. The assay mode was RATE-A. The reagent blank value was Expression and purification of recombinant AAO. obtained with distilled water. Samples were prepared by E. coli cells harboring pTV118N/AAO were cultivated adding PE to the plasma. The fresh human plasma was for 20 h at 30 C in 20 liters medium containing 3.0% collected from consenting healthy donors, pooled, and yeast extract, 3.0% sucrose, and 50 mg/ml ampicillin at then frozen just before assay. pH 6.8, after which expression was induced by the addition of 1 mM isopropyl--d-thiogalactopyranoside to Results and Discussion the medium. AAO-expressing cells were subsequently harvestedAdvance by centrifugation, suspended in 20 View mM Tris– Production of amine oxidase from Arthrobacter sp. HCl (pH 8.0), and then disrupted by ultrasonication on Most copper amine oxidases are inducible enzymes- ice. After removing the cell debris by centrifugation at e.g., A. globiformis expresses two separately inducible 15;000 g for 20 min, the supernatant was loaded onto a copper amine oxidases: phenylethylamine oxidase Q Sepharose Big Beads BPG100 column (2 liters, (AGAO)12) and histamine oxidase.13) In the present GE Healthcare) pre-equilibrated with 10 mM Tris–HCl study, AAO was also inducible, and little activity was buffer (pH 8.0). The column was then washed with the detected unless phenylethylamine was present in the same buffer, followed by washing with 0.2 column medium. Arthrobacter sp. was cultivated in the enriched volumes of 2 mM CuCl2. The protein was then eluted medium, and the absorbance at 660 nm reached 23.1 with a linear gradient of 0 to 0.5 M KCl in the same after 40 h of cultivation.Proofs AAO from Arthrobacter sp. buffer. The fractions containing AAO were collected, was purified about 15-fold to homogeneity on SDS– and solid KCl was added to a concentration of 3 M, after PAGE, with a yield of 54% (Fig. 1 and Table 1). which the protein solution was loaded onto a Phenyl Sepharose 6 Fast Flow XK50 column (0.5 liters, GE Biochemical characteristics of AAO Healthcare) pre-equilibrated with the same buffer. After The biochemical properties of AAO are summarized the column was washed with the same buffer, the protein in Table 2. The protein had a native molecular mass of was eluted with a linear gradient of 3 to 0 M KCl. The 140 kDa and a subunit size of 71 kDa, indicating that it active fractions were pooled, concentrated using a was a typical copper amine oxidase with respect to both 30-kDa centrifugal filter device, and desalted by passage its subunit size and its homodimeric structure. The through a Sephadex G-25 Superfine column (5 50 cm, activity of AAO increased as the temperature was GE Healthcare) pre-equilibrated with 20 mM Tris–HCl increased from 17 C to approximately 45 C. At higher (pH 7.5). The entire operation was carried out at room temperatures the activity declined, probably due to temperature (about 25 C). inactivation of the enzyme. The temperature optimum was 42 C, comparable to that of monoamine oxidase Preliminary study of the phosphatidylethanolamine from E. coli K-12.14) Arrhenius plots for kidney diamine assay. Two separated reaction mixtures were used in the present PE assay. Reaction mixture #1 was for elimi- nating biological materials that might be substrates of Table 1. Purification of Amine Oxidase from Arthrobacter sp. AAO in the samples. These interfering substances are Total activity Protein Specific activity Yield Step oxidized by AAO, and generated H2O2 reacts with N- (kU) (g) (U/mg) (%) ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS, Cell free extract 37 44 0.8 100 Dojin, Kumamoto, Japan) through peroxidase activity to (NH ) SO 4 2 4 35 15 2.4 96 form colorless substances. Reaction mixture #1 con- precipitation tained 100 mM Tris–HCl pH 7.5, 3 U/ml AAO, 5 U/ml DEAE sep. FF 22 2.0 11.5 61 Q sep. HP 20 1.6 12.7 54 peroxidase, 0.3% Triton X-100, and 1.79 mM TOOS. Reaction mixture #2 was for hydrolysis of the phospho- The cell extract was prepared from 20 liters of culture. 80365-4 H. OTA et al.

A B Table 3. Kinetic Parameters of the Amine Oxidase from Arthro- bacter sp. kDa 1423 Vmax Km Kcat/Km 97.2 Substrate (mmol/min/mg) (mM)(s1 mM1) 66.4 Phenylethylamine 7.8 0.003 2,962 45.0 Tyramine 22 0.017 1,481 Dopamine 15 0.23 76 29.0 Amylamine 11 0.20 67 20.1 Histamine 10 0.48 25 Hexylamine 12 0.10 17 14.3 Agmatine 0.7 0.26 3.2 Arylamine 1.6 0.13 1.8 Fig. 1. SDS–PAGE Analysis of Amine Oxidase from Arthrobacter sp. Ethanolamine 9.0 15 0.7 A, Purified amine oxidase from Arthrobacter sp. B, Crude extract The apparent kinetic parameters were examined under standard assay of amine oxidase heterologously expressed in E. coli strains JM109, conditions. The substrate concentrations were as follows: phenylethylamine, JM101, LE392, and TH2 at 30 C were loaded onto lanes 1 to 4. The 0.017–0.033 mM; tyramine, 0.02–0.033 mM; dopamine, 0.01–0.20 mM; amyl- arrow indicates the position of amine oxidase. amine, 0.05–2.0 mM; histamine, 0.05–0.50 mM; hexylamine, 0.05–1.0 mM; agmatine, 0.25–5.0 mM; arylamine 0.05–5.0 mM; ethanolamine, 2.5–20 mM. Table 2. Biochemical Properties of Amine Oxidase from Arthro- bacter sp. Substrate specificity and kinetics of AAO Parameter The reactivities of tyramine, monoethanolamine Molecular mass (ethanolamine), trimethylamine, triethanolamine, gluta- Native enzyme (kDa)a 140 mine, ethanediamine, dopamine, histamine, propanol- AdvanceCalculated (Da) View71,087 amine, agmatine, amylamine, methylamine, phenyle- Arrhenius activation energyb thylamine, propylamine, arylamine, benzylamine, (kJ/mol, 17–37 C) 60 hexylamine, 1,4-diaminobutane, and cadaverine (all at pH optimumc 6.5–7.5 pH stabilityd 5.5–8.5 1mM) were tested. Among these, AAO showed activity Thermostabilitye about 50 C toward tyramine, ethanolamine, dopamine, histamine, agmatine, amylamine, phenylethylamine, arylamine, and aThe molecular mass of the native enzyme was analyzed by gel filtration on a TSK gel column G3000SWXL (Tosoh, Tokyo) pre-equilibrated with 50 mM hexylamine, but little or no activity toward the others. potassium phosphate buffer (pH 7) containing 0.2 M NaCl. The flow rate was The results of our analysis of the enzyme kinetics with 0.4 ml/min. The marker proteins used ranged from 12.4 to 240 kDa (Oriental each of the nine active substrates are summarized in Yeast, Osaka, Japan). bActivation energy was calculated from the slope of the linear part of the Table 3. We found that for the substrate concentrations Arrhenius plot (17–37 C; complete range, 17–52 C). Proofs indicated in the table, the reactions all followed c The pH optima were determined using citrate–NaOH (pH 4.5–6.0), Michaelis-Menten kinetics. Beyond those concentra- potassium phosphate (pH 6.0–7.5), Tris–HCl (pH 7.0–9.0), and glycine– NaOH (pH 9.5–11.0) buffers under standard assay conditions. tions, substrate inhibition was observed. While the dAliquots of 0.5 U/ml in the buffer systems (100 mM) used to determine the Vmax for phenylethylamine was smaller than that for pH optima were incubated for 3 h at 37 C. After incubation, the remaining tyramine, dopamine, amylamine, histamine, hexyl- activities were analyzed. eAliquots of 1.0 mg enzyme/ml in 20 mM potassium phosphate (pH 7.0) amine, or ethanolamine, phenylethylamine had a much were incubated in sealed tubes for 30 min at 0–80 C. After incubation, the lower Km, indicating that it is the most preferred tubes were rapidly cooled in an ice bath and analyzed for activity. substrate, with the highest kcat/Km. The kcat/Km value for tyramine was about half that for phenylethylamine, and dopamine, amylamine, histamine, and hexylamine oxidase show two-phase linearity with a transition point had much smaller kcat/Km values (approximately 0.6– at about 40 C,15) which is generally interpreted as being 2.6% of the phenylethylamine kcat/Km value). Thus the due to a change in conformational flexibility. By substrate specificity of AAO indicated that it is to be contrast, those of AAO from Arthrobacter sp. showed phenylethylamine oxidase. But AAO differs from a linearity over a temperature range from 17 Cto37C, previously purified and characterized phenylethylamine and an activation energy of 60 kJ/mol was calculated oxidase from A. globiformis (AGAO):8,12,13) whereas from the slope. The pH optimum of the enzyme was AGAO shows no activity toward histamine,14) AAO about pH 7, comparable to that of monoamine oxidase showed a relatively high Vmax (10 mmol/min/mg) with from Sarcina lutea,16) and pH stability was in the range histamine. We found the Km toward ethanolamine of of pH 5.5–8.5. In 20 mM potassium phosphate (pH 7.0), AAO to be 15 mM. Although this value is higher than AAO retained all of its activity after 30 min at 40 C, but that for the other substrates tested, it is sufficient for the it lost 60% of its activity at 60 C, and was completely rate assay of PE, since the PE concentration in human inactivated by incubation at 70 C. Although the enzyme plasma is reported to be 9:2 2:2 mg/dl.17) The sub- had a neutral pH optimum, it was stable over a wide strate specificities for ethanolamine of amine oxidases range of pHs, which is advantageous in a diagnostic from A. globiformis, Arthrobacter strain P1, S. lutea, reagent. E. coli K-12, and Aspergillus niger AKU 3302, have not Amine Oxidase from Arthrobacter sp. 80365-5 been examined yet.12,14,16,18,19) On the other hand, an Table 4. Effects of Monoamine Oxidase Inhibitors on the Activity of ethanolamine oxidase (EC 1. 4. 3. 8) from Arthrobacter Amine Oxidase from Arthrobacter sp. 20) was reported in 1964. This enzyme might be an amine Inhibitor (0.1 mM) Relative activity (%) oxidase as is AAO in view of characteristics similar to Hydroxylamine 1 AAO, including the Km for ethanolamine (5 mM) and Isoniazid 2 isoniazid sensitivity. Clorgyline 99 Pargyline 99 Inhibition by specific monoamine oxidase inhibitors Semicarbazide 0

Although classification of amine oxidases based on Aliquots of the enzyme (0.5 U/ml in the 20 mM potassium phosphate specific inhibitors might be somewhat of an oversimpli- pH 7.0) were incubated with the inhibitors for 1 h at 37 C, and then the fication, it is useful in some instances. The classification activities were analyzed. of flavin containing amine oxidoreductase (MAO) was primarily based on specific inhibition of MAO-A by clorgyline and of MAO-B by deprenyl and by pargy- the AAO gene includes 1,923 bp encoding 641 amino line.21) Semicarbazide-sensitive amine oxidase, a copper acids with a calculated molecular weight of 71,087 amine oxidase, is not inhibited by clorgyline, depreny, or (DDBJ accession no. AB438107), which corresponds to pargyline, though it is almost completely inhibited by the subunit molecular mass of about 70 kDa as deter- semicarbazide.22) Similarly, AAO activity was markedly mined by SDS–PAGE. When we carried out a BLASTP inhibited by pretreatment with carbonyl reagents, in- search for the deduced amino acid sequence of AAO, we cluding semicarbazide (Table 4). By contrast, clorgyline found that the sequence showed a high degree of identity and pargyline had no effect on its activity. These findings with Cu-containing amine oxidases, including AGAO are in good agreement with earlier reports on semi- (U03517, 63%),12) histamine oxidase from A. globifor- carbazide-sensitiveAdvance amine oxidases.22,23) Viewmis (D38508, 58%),13) and methylamine oxidase from Arthrobacter sp. strain P1 (L12983, 44%).18) Because Nucleotide sequences and amino acid sequence the tertiary structures of both the holo- and the apo- alignment forms of AGAO have analyzed cystallographically,8) we The N-terminal amino acid sequence of AAO is next compared the deduced amino acid sequence of shown in Fig. 2. The complete nucleotide sequence of AAO with that of AGAO (Fig. 2). An active site Proofs

Fig. 2. Alignment of the Amino Acid Sequences of Amine Oxidase from Arthrobacter sp. (AAO) and A. globiformis (AGAO, PDB 1AVK). Partial peptide sequences determined by automated Edman are underlined. The regions used to design the PCR primers are double-underlined. Asterisks indicate residues common to both enzymes. The consensus sequence N-Y-D-Y is highlighted by a black box with white letters. The amino acid residues assumed to be involved in binding copper are boxed. 80365-6 H. OTA et al. tyrosine side chain of AGAO is post-translationally 80 modified in a self-processing reaction to form TPQ.5,6) The position of the TPQ cofactor has been determined to 60 be Y382 in AGAO,8) and the corresponding amino acid is Y377 in AAO. The tetrapeptide consensus sequence NY(D/Q)Y, within which the first tyrosine residue is 40 converted to the TPQ, was also conserved in AAO mAbs/min 19) (Fig. 2). In AGAO, the Cu atom is coordinated by 20 three His residues,8) and these, too, were conserved in AAO, as H424, H426, and H585 (Fig. 2). Taken together, these results suggest that the three-dimensional 0 structure and active site of AAO are similar to those 020406080 of AGAO. Phosphatidylethanolamine (mg/dl)

Heterologous expression of AAO in E. coli Fig. 3. Calibration Curve for Phosphatidylethanolamine Assay It has been reported that although recombinant AGAO Utilizing the Amine Oxidase from Arthrobacter sp., Phospholipase D, and Peroxidase. was successfully expressed using a pET system, hista- The regression line was defined by the equation Y ¼ 0:983X þ mine oxidase was mainly produced in an insoluble form 0:17, and R2 ¼ 0:999. when either pET or pTV118 plasmid was used, but soluble expression was accomplished using pTrc99A.13) In the present study, the yield of AAO protein was also evaluated interference by these potential biological dependent on the host strain. SDS–PAGE revealed the materials, but, no effects were found. This method was presenceAdvance of only low levels of a 70-kDa polypeptide Viewalso free from interference by EDTA, an anticoagulant when E. coli strain JM101, LE392, or TH2 was trans- frequently used, up to 300 mg/dl. AAO thus appear to formed, and strain W3110 transformants harboring be potentially useful tools for the accurate determination pTV118N/AAO barely grew on LB medium, even of PE in plasma. without Cu2þ in the medium. By contrast, when strain JM109 was used, the 70-kDa protein accounted for In summary, we developed an efficient protocol for approximately 40% of the total soluble protein of the the high-level production of an AAO with ethanolamine cells (Fig. 1). The heterologously overexpressed AAO oxidase activity. This enzyme was found to be a in E. coli JM109 was in an inactive form due to the semicarbazide-sensitive Cu-containing amine oxidase, absence of Cu2þ in the medium. Consistently with its and its preferredProofs substrate was phenylethylamine. We inactivation, the AAO protein bound to the Q Sepharose also developed a novel, simple PE assay system that can Big Beads during the first purification step was initially be applied to an automatic clinical analyzer based on colorless. As the column was washed with 2 mM CuCl2, AAO ability to oxidize ethanolamine. Further develop- however, the bound protein gradually turned brownish ment of a convenient and practical PE assay method is pink, and was eluted as an active form, which confirmed currently under way. that the enzyme was a Cu-containing amine oxidase. On purification, the purity of the recombinant enzyme was References more than 90% as judged by SDS–PAGE analysis, and the enzyme had a specific activity of 11 U/mg; more- 1) Vance, J. E., Phosphatidylserine and phosphatidyletha- nolamine in mammalian cells: two metabolically-related over, the properties of the recombinant AAO were aminophospholipids. J. Lipid Res., 49, 1377–1387 identical to those of the wild-type enzyme. (2008). 2) Cole, D. E., Farag, S., and Dooley, K. C., Ethanolami- PE assay nuria: a non-specific laboratory finding in the seriously Finally, to confirm the practical feasibility of applying ill infant. Clin. Biochem., 21, 297–300 (1988). the enzyme to PE assay, we examined the following 3) Creer, M. H., Pastor, C., Corr, P. B., Gross, R. W., and using two reaction mixtures: within-run, recovery, Sobel, B. E., Quantification of choline and ethanolamine calibration curve, minimum detection limit, and inter- phospholipids in rabbit myocardium. Anal. Biochem., fering substances. The within-run (n ¼ 20) coefficient of 144, 65–74 (1985). variation was 2.17% for 15.4 mg/dl of PE. The additive 4) Imamura, S., and Horiuti, Y., Enzymatic determination recoveries were 96 and 97% for 5 and 10 mg/dl addition of phospholipase D activity with choline oxidase. J. Biochem., 83, 677–680 (1978). of PE to the plasma. 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