Agric. Biol. Chem., 53 (6), 1671 -1677, 1989 1671

Properties of a NewEnzyme, Oxidase, from Pseudomonas maltophilia LB-86 Yoshikazu Isono, TomokoSudo and MasamiHoshino Biwako Research Laboratory, Otsuka Foods Co., Ltd., Karasaki, Otsu, Shiga 520-01, Japan Received January 23, 1989

Nucleoside oxidase purified from Pseudomonas maltophilia LB-86 had mol. wt. = 130.000 and was composed of one each of four non-identical subunits: subunit a, 76,000; subunit /?, 33,000; subunit y, 18,000; subunit <5, 14,000. The enzyme contains 1 mol of covalently bound FAD, 2g atoms of non- heme iron, 2 mol of labile sulfides, and 1 mol of heme per mol protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390nm, and shoulders at 343 and 450nm. The enzymecatalyzes the oxidation of various , and the Kmvalue for was 4.4 x 10"5 M. The enzyme was most active at pH 5-6, and was most stable between pH 5.0-6.0 and at temperatures below 60°C. The activity was strongly inhibited by /V-bromosuccinimide and potassium cyanide.

As reported previously,1} a new enzyme oxidase was prepared from Pseudomonas maltophilia LB- catalyzed the oxidation of nucleosides without 86, as described previously.2' a requirement for an exogenous co factor. This nucleoside-oxidizing enzyme was quite dif- Assay of nucleoside oxidase activity. Enzymeactivity was assayed by measurementof oxygen uptake with an ferent from any previously reported , Oxygraph model 8 oxygen electrode (Central Kagaku Co., and thus we have designated tentatively it as Ltd.) placed in a thermostatically controlled vessel at "nucleoside oxidase." Nucleoside oxidase was 25°C. The reaction mixture contained 7jumol of inosine, 100/miol of potassium phosphate buffer (pH 6.0), and a purified from the cell-extract of Pseudomonas suitable amount of nucleoside oxidase in a final volume of maltophilia LB-86 by ammoniumsulfate frac- 1.0ml. One unit of enzyme activity was defined as the tionation, heat treatment, columnchramatog- amount of enzyme that consume 1jimo\ of oxygen raphy on DEAE-Toyopearl, and gel filtration per min. twice on a Sephacry S-200 column.2) The purified enzyme preparation was homo- Measurement of protein. Protein was measured by the geneous on polyacrylamide gel electro- method of Lowry et al.,3) with crystalline bovine serum albumin as the standard. phoresis. This paper describes molecular and catalytic properties of the enzyme. Measurementof molecular weight. Molecular weight was measured by gel filtration on a column (2.2 x 79.5cm) of Sephacryl S-200 by the method of Andrews.4' Elution was Materials and Methods done with 50mMpotassium phosphate buffer, pH 7.4, containing 0.5m NaCl. The low rate was adjusted to 10ml Materials. Sephacryl S-200 was obtained from cm^hr"1, and 3.0ml fractions were collected. Five pro- Pharmacia Fine Chemicals, Sweden. Phosphodiesterase tein standards were used: bovine liver catalase (mol. wt. was purchased from Sigma Chemical Co., U.S.A. Other 232,000), rabbit muscle aldolase (mol. wt. 158,000), bovine chemicals were of analytical grade available from com- serum albumin (mol. wt. 67,000), hen egg albumin (mol. mercial sources. wt. 45,000), and bovine pancreas chymotrypsinogen A (mol. wt. 25,000). Vo was measured with Blue Dextran Preparation of nucleoside oxidase. Purified nucleoside 2,000. The molecular weights of the subunits were esti- Abbreviations: MES, 2-(A^-morpholino) ethanesulfonic acid; SDS, sodium dodecyl sulfate. 1672 Y. Isono, T. Sudo and M. Hoshino mated by sodium dodecyl sulfate (SDS)-acrylamide gel 5C4-300 column (04.5 x 150mm, Nacalai Tesque Inc.). electrophoresis by the method of Weber and Osborn.5) Subunis were eluted by a gradient of acetonitrile from 0 to The enzyme was denatured by treatment with 1%SDS 72% in 0.05% trifluroacetate solution. solution containing 1%2-mercaptoethanol at 100°C, lO min. Standard proteins: horse heart c (mol. AMPassay. AMPwas assayed by HPLCunder the wt. 12,400), bovine pancreas chymotrypsinogen A (mol. following conditions: column, Fine SIL C18 (Japan wt. 25,000), bovine erythrocyte carbonic anhydrase (mol. Spectroscopic Co., Ltd. 04.5 x250mm); solvent, 10mM wt. 29,000), hen egg albumin (mol. wt. 45,000), bovine KH2PO4-H3PO4 (pH 3.0)/methanol (18 : 1, by volume); plasma albumin (mol. wt. 67,000), rabbit muscle phospho- flow rate, 1.5ml/min and detection, UVat 250nm. The rylase B (mol. wt. 97,400), and E. coli /?-galactosidase chromatogramwas recorded on an intergrator, and AMP (mol. wt. 116,000). was quantified on the basis of peak area using calibration Isoelectricfocusing. Isoelectric focusing was done at 4°C for 48hr on an LKB column (110ml) containing 1% Results Carrier Ampholite of pH 3.5 x 10.0 (LKB-Produkter AB, Sweden) by the method of Vesterberg and Svensson.6' Molecular properties of nucleoside oxidase Spectrophotometric measurements. Spectrophotometric 1) Molecular weight and subunit structure. As measurements were done with a Shimadzu model UV-240 shown in Fig. 1, the molecular weight of the recording spectrophotometer. enzyme was 130,000 by Andrew's gel filtration method on Sephacryl S-200. The subunit struc- Measurementof metals. Total iron was measured using 1,10-phenanthroline and atomic absorption with a Hitachi ture was analyzed by SDS-gel electrophoresis. atomic absorption spectrophotometer, model 180-50. There were four bands of stained protein, Non-heme iron was extracted with trichloroacetic acid, designated tentatively as subunits a, /?, y, and and the liberated iron was measured as ferrous 1,10- 3. By calibration with several standard pro- phenanthroline complex, as described by Massey8) teins, their molecular weights were 76,000, (Method A). Non-heme iron was also extracted into 33,000, 18,000, and 14,000. ethanol after reduction with sodium dithionite and was afterwards measured with 4,7-diphenyl-1,10-phenan- The purified enzymeprotein was also ana- throline as described by Doeg and Ziegler9) (Method lyzed by reverse-phase HPLCusing a Cosmosil B). 5Q-300. Figure 2 shows typical elution pat- Other metal contents of nucleoside oxidase were mea- terns of the enzyme protein at pH 7.0, 3.4, and sured by atomic absorption. Analytical grade metal salts wereused as standards.

Measurement of labile sulfide. Labile sulfide was esti- mated by the method of King and Morris10' with the following minor modifications. A sample of nucleoside oxidase was made up to a volume of0.7ml by the addition of water in a centrifuge tube with ajoint stopper. To this, 0.5 ml of alkaline zinc reagent (freshly prepared by adding 5 volumes of2.6% zinc acetate to 1 volume of6% NaOH) was added. After the tube was stoppered, and shaken for 1 min, 0.1 ml of 0.02m A^N'-dimethyl-p-phenylendiamine sulfatein 7.2n HC1and0.1 ml of0.03m FeCl3 in 1.2n HC1 were added to the tube in rapid succession. After 1 hr, 1.0ml of water was added, and the precipitate was re- moved by centrifugation. The color of the supernatant Fig. 1. Measurement of the Molecular Weight of solution was measured at 670nm against a reagent blank. Standard solutions of sodium sulfide were prepared as Nucleoside Oxidase by Filtration on Sephacryl S-200. described by King and Morris.10' The enzyme (Z) and protein standards (A, bovine liver catalase; B, rabbit muscle aldolase; C, bovine serum albumin; D, hen egg albumin; and E, bovine pancreas Preparations ofsubunits a,p,y, and 3. Subunits a, y, and p-S complex were prepared by reverse-phase HPLCusing chymotrypsinogen A) were chromatographed at 4 °C on a two chromatography pumps, Waters model 6000Aand column (2.2 x 79.5 cm) equilibrated with 50 mMphosphate Waters model 660, a solvent programmerwith a Cosmocil buffer, pH 7.5, containing 0.5m NaCl. Properties of Nucleoside Oxidase 1673

Fig. 3. Gel Permeation HPLCofDenatured Peak (a) on TSKgel G3000SW XL. Peak (a) was denatured by treatment with 1% SDS solution containing 0.05m dithiothreitol at 100°C, for lOmin, and put on a column of TSKgel G3000SWXL. Chromatographic conditions are as follows: eluent, 0.2m sodium phosphate buffer, pH 7.0, containing 0.2% SDS; flow rate, l.Oml/min; detection, absorbance at 280nm.

Fig. 2. Reverse-phase HPLCof Nucleoside Oxidase on Cosmosil 5Q-300. Theenzymesolution wasput on a columnof Cosmosi] 5C4-300 (04.5 x 150mm). Elution (l.Oml/min) was done with a gradient ofacetonitrile from 0 to 72%in (1) 10mM phosphate buffer, pH 7.0; (2) 10mMphosphate buffer, pH 3.4; (3) 0.05% trifluoroacetate, pH 2.4.

2.4. At pH 7.0, the enzyme was eluted as a single peak, and this peak had the activity. Whenthe enzyme was eluted at pH 3,4, the peak that had the activity decreased with the appearance of three additional peaks, desig- nated (a), (b), and (c), and these three peaks had no activity. At pH 2.4 the peak of native Fig. 4. Absorption Spectra of Nucleoside Oxidase. enzyme protein disappeared and three peaks The protein concentration was 0.48mg per ml in 20mM remained. On these three chromatographies, MES buffer, pH 6.0, ( ), oxidized form; ( ), reduced form treated with sodium hydrosulfite; ( -.), the protein recoveries were almost 100%. The reduced form treated with inosine. The inset represents a relative ratio of peak area or peak ptotein was differential spectrum of the oxidized and inosine reduced 1.00 (a): 1.52 (b): 0.45 (c) or 1.00 (a): 1.66 (b): forms. 0.43 (c). These three peaks were electropho- resed on SDS gel. Peak (a) had two protein linked together by disulfide bonds as indicated bands identified as subunits f$ and (5, and peak by the following results. When peak (a) was (b) and (c) were identified as subunits a and y, denatured by treatment with 1 %SDSsolution, respectivey. Subunits fi and S appear to be but without 2-mercaptoethanol, and elec- 1674 Y. Isono, T. Sudo and M. Hoshino trophoresed, a single band of protein was unit a showed maxima at 450nm, 343nm, and obtained. Peak (a) denatured by treatment 275nm, and minima at 392nm, 315nm, and with 1% SDS solution containing 0.05m di- 249nm (Fig. 5). This is typical for a flavo- thiothreitol at 100°C, for lOmin, was used in protein. Onthe basis of the molar extinction gel permeation HPLC using TSK gel G- coefficient for free FAD at 450nm (e450nm= 3000SW XL (Tohso Co., Ltd.). Two peaks ll.3mM"1cm~1) a flavin-protein (subunit a) corresponding to subunits /? and d were eluted ratio of 1 was calculated. In the other sub- with a yield of almost 100% (Fig. 3). The units, flavin was not detected. Treatment of relative ratio of peak areas or peak proteins the native enzyme with 5% trichloroacetic was 1.00 (/?): 0.38 (5) or 1.00 (jS): 0.46 (<5). acid for 1hr at 4°C, or heating at 100°C re- These results suggest the molar ratio ofa: /?: y: sulted in a supernatant without any absor- dwas 1 : 1 : 1 : 1,andtheenzymeconsistoffour bance in the visible range. This shows the pres- non-identical subunits. ence of a covalently bound flavin in the a 2) Absorption spectrum. Absorption spec- subunit. To discriminate between FMNand trum of the purified enzyme had maxima at FAD,an attempt was made to measure AMP 390nm and 278nm, and shoulders at 343nm and 450 nm (Fig. 4). The extinction coefficient (E\°^) of nucleoside oxidase at 278nm in Table I. Identification of FADby Liberation of AMP 20mM MES buffer of pH 6.0 was 14.7. The enzyme was reduced by inosine, the substrate, The enzyme, AMP, and FAD each in 0.5 ml 0.2M Tris- HC1 buffer, pH 8.5, were incubated with 0.3 mg oftrypsin or by sodium hydrosulflte as also shown in the at 37°C for 3 hr. The heat-inactivated digest was treated absorption spectum: reductions were rever- with 5 fig phosphodiesterase for 30min, and then AMP sible. A differential spectrum of oxidized and was assayed (see Materials and Methods). inosine-reduce forms showed maxima at Substrate Substrate added AMP recovered 430nm and 626nm, and minima at 480nm and around 340nm. AMP 12.3 (nmol) 12.1 (nmol) 3) Cofactors. FAD 18.6 18.4 Nucleoside oxidase 6.5 5.8 (a) Flavin. The absorption spectrum of sub-

Table II. Metal and Labile Sulfide Contents of Nucleoside Oxidase

ComponentF, JandContentanalytical procedure Total iron (1,10-Phenanthroline method) 2.7 (Atomic absorption) (g atms/mol2.8 enzyme) Non-heme iron (Method A) 1.8 (Method B) 1.7 Copper (Atomic absorption) 0 Zinc (Atomic absorption) 0 Molybdenum(Atomic absorption) 0 Manganese (Atomic absorption) 0 Labile sulfide 1.9 Fig. 5. Absorption Spectra of Subunit a. (mol/mol enzyme) The protein concentration of the sample was 0.55mg perml. A. oxidized form; B, reduced form after addition of Molecular weight = 1 30,000 was used for calculation See sodium hydrosulfite. Materials and Methods. Properties of Nucleoside Oxidase 1 675 after treatment of the purified enzyme with component. venomphosphodiesterase. A tryptic digestion 4) Isoelectricpoint. Isoelectric focusing (on a was done before incubation with phosphodies- column containing Carrier Ampholite at pH terase. After heat inactivation, AMPwas 3.5-10.0) showed that the pi value of the found in the supernatant (see Materials and enzymewas 5.3. Methods) in amounts equivalent to the flavin content of the treated enzyme(Table I). Thus Enzymatic properties of nucleoside oxidase nucleoside oxidase contains FAD covalently 1) Substrate specificity. The ability of the bound to a polypeptide chain. enzyme to catalyze the oxidation of various (b) Iron and labile sulfide. Contents of iron nucleosides and related compounds was in- and labile sulfide were measured. The results vestigated as shown in Table III. Various are summarizedin Table II. Analyses of the nucleosides were oxidized rapidly while nu- enzymepreparations by the atomic absorption cleotides, bases, and ribose were not oxidized and 1,10-phenanthroline methods7) showed 3 g at all. The Michaelis constant {Km) was atoms of total iron permol enzyme. Copper, 4.4x 10~5M for inosine from a Lineweaver- molybdeum, zinc, and manganese were not Table III. Substrate Specificity of detected in the enzyme. Non-hemeiron of the Nucleoside Oxidase enzymewas estimated to be 2g atoms permol Nucleoside oxidase was incubated with each substrate of enzyme by the methods described in under standard assay conditions. Materials and Methods, and analysis of the enzyme for labile sulfide yielded 2mol of sul- Substrate_, , Concentration. Relative/0/. activity fide permol of enzyme. In pyridine-NaOH W (%) solution, the enzyme reduced by sodium hy- Inosine 10 100 drosulfite had the spectrum shown in Fig. 6, Adenosine 1 0 97.3 with maxima at 413nm, 520nm, and 549nm. Guanosine 5 121 Xanthosine 1 0 1 25 This indicates that the enzymecontains a heme Uridine 1 0 92.8 Cytidine 10 77.5 Deoxyinosine 1 0 80.7 Deoxyadenosine 1 0 8 1.4 Deoxyguanosine 1 0 92.3 Deoxycytidine 1 0 77. 3 Thymidine 1 0 55.8 D-Ribose 20 0 D-Ribose-1 -P 20 0 D-Ribose-5-P 20 0 2-Deoxy-d-ribose 20 0 Hypoxanthine 5.2 0 Xanthine 0.4 0 Adenine 10 0 Guanine 0.4 0 Thymine 20 0 Cytosine 20 0 Uracil 20 0 5 -IMP 10 0 5 -IDP 10 0 5 -GMP 10 0 5 -AMP 10 0 Fig. 6. Absorption Spectrum of Reduced Nucleoside 5 -ADP 10 0 5 -ATP 10 0 Oxidase in Pyridine-NaOH Solution. 2'(3')-AMP 10 0 To 1.5ml ofenzyme (1.56mg of protein) was added a few 2 (3 )-IMP 10 0 crystals of sodium hydrosulfite, 0.5ml of pyridine, and 2'(3')-UMP 10 0 0.2ml of2N NaOH. 1676 Y. Isono, T. Sudo and M. Hoshino

Table IV. Effect of Inhibitors on Nucleoside Oxidase Activity Nucleoside oxidase (0.02unit) was incubated with in- osine in the presence of inhibitors under the standard assay conditions. ,.,. Concentration Relative activity Inhlbltor M (%)

None 100 Potassium cyanide 1 34.8 10 0 Fig. 7. Effects of pH on Enzyme Activity. Sodium azide 1 81.5 The enzyme activity was measured with inosine as a 10 45.6 EDTA 1 101 substrate in the following buffer: -O-, potassium phos- o-Phenanthroline 1 94. 7 phate buffer (^=0.05); -#-, acetate buffer (/i=0.05). a,a'-Dipyridyl 1 96.7 Quinacrine 1 1 00 Hydrazine sulfate 1 101 Iodoacetic acid 1 1 00 /7-Chloromercuri- 1 98.9 benzoic acid Phenylmethyl- 1 95. 3 sulfonyl fluoride HgCl2 1 69.2 Pb(CH3COO)2 1 81.3 jV-Bromosuccinimide 1 0

given in Table IV. The enzyme activity was inhibited by potassium cyanide and sodium Fig. 8. Effects of pH and Temperature on the Stability. azide, commoninhibitors of iron porphyrine A: The enzyme was kept at 37°C for 60min at various enzymes. The enzyme also lost considerable pHs, and then the remaining activity was measured. activity on modification with A^-bromosuc- B: The enzyme in 20mMMESbuffer (pH 6.0) was kept at cinimide, which is highly reactive with trypto- various temperatures for 15 min, and then the remaining phan residues. activity was measured by the standard assay method. Discussion Burk plot. 2) Effects ofpH on the activity. The maximal Nucleoside oxidase, a novel nucleoside- enzyme activity was observed in the range of oxidizing enzyme, was found in a few bacterial pHs 5~6, when inosine was used as a sub- strains and was purified to apparent homo- strate (Fig. 7). geneity from the cell-free extract of Pseu- 3) Effects of pHand temperature on the domonas maltophilia LB-86. Nucleoside oxi- stability. The enzyme was most stable from pH dase had a molecular weight of 130,000, and 5 to 6when stored 37°C for 1 hr (Fig. 8A). The contained four non-identical subunits, a, /?, enzymewasincubated at various temperatures y, and 3. The molecular weights estimated for 15min and the remaining activities were by SDS gel electrophoresis were 76,000, assayed at 25°C. The enzyme was stable at 33,000, 18,000, and 14,000 for subunits a, j3, y, temperature below 60°C and completely lost and (5, respectively. The molar ratio ofa: /?: y: 5 its activity at 75°C (Fig. 8B). was calculated to be about 1:1:1:1. As- 4) Effect ofinhibitors. The effects of a num- suming a 1:1:1:1 ratio of the subunits in ber of inhibitors on the enzyme reaction are the native enzyme, a molecular weight of Properties of Nucleoside Oxidase 1677

141,000 was obtained for the native enzyme, azide, common inhibitors of heme enzymes, which is within 10% of the value obtained by also support the participation of hemein the gel filtration. These results show that the en- oxidation process. zyme is composed of four different kinds of Thus, nucleoside oxidase is considered to be subunits. Several other enzymesll)12) that are an iron-sulfur hemoflavo protein. However, the composed of three or four non-identical sub- investigation on the participation of these units have been reported, but these are components in catalytic activity and their se- unusual. quence in the electron transfer pathway, were The spectrum of subunit a included peaks at not done in this study. 343nm and 450nm. The peak at 450nm disap- The enzyme was active on various nucle- peared upon reduction with sodium hydrosul- osides, and nucleoside and ribonucle- fite. These spectral characteristics indicate that osides were oxidized somewhat more rapidly subunit a is a flavoprotein. The flavin was than nucleosides and deoxyribo- covalently bound to subunit a, and the molar nucleosides. On the other hand, various nu- ratio offlavin: subunit a was calculated to be 1. cleotides, bases, and ribose were not oxidized The flavin was identified as FAD by HPLC at all. Nucleoside oxidase is stable and has a after digestion with trypsin and phosphodies- high structural specificity toward nucleosides. terase. This type of linkage was first reported These characteristics allow the enzyme to be for succinate dehydrogenase of bovine heart used for measurement of nucleosides. mitochondria,13) and several oxidase12'14^17) that contain covalently bound FADwere re- References ported. Sarcosine oxidase12) from Corynebac- 1) Y. Isono, M. Hoshino and T. Sudo, Agric. Biol. terium sp. has been shown to contain 1mol Chem., 52, 2135 (1988). of covalently bound FADand 1 mol of non- 2) Y. Isono, T. Sudo and M. Hoshino, Agric. Biol. covalently bount FAD permol of enzyme. Chem., 53, 1663 (1989). Non-covalently bound flavin was not detected 3) O. H. Lowry, N. J. Rosebrough, A. L. Farrand R. J. in nucleoside oxidase, and covalently bound Randall, J. Biol. Chem., 193, 265 (1951). flavin was found only in subunit a. There- 4) P. Andrews, Biochem. J., 91, 222 (1964). 5) K. Weber and M. Osborn, J. Biol. Chem., 244, 4406 fore, nucleoside oxidase contains only 1 mol (1969). of FADper mol ofenzyme. 6) O. Vesterberg and H. Svensson, Ada. Chem. Scand., Wheniron not bound to hemewas measur- 20, 820 (1966). 7) M. Van de Bogart and H. Binert, Anal. Biochem., 20, ed, it was clear that 2g atoms of iron permol 325 (1967). of enzyme exist as non-heme iron which is 8) U. Massey, J. Biol. Chem., 229, 763 (1957). extractable with 5% trichloroacetic acid or 9) K. A. Doeg and D. M. Ziegler, Arch. Biochem. ethanol after sodium hydrosulfite. Analysis Biophys., 97, 37 (1962). of the enzyme for labile sulfide yielded 2 mol of 10) T. E. King and R. O. Morris, Methods Enzymol, 10, sulfide permol of enzyme. These results sug- 634 (1967). ll) J. N. Ihle and L. S. Dure, J. Biol. Chem., 247, 5034 gest the presence of iron-sulfur center. Onthe (1972). other hand, analyses of the purified enzyme 12) M. Suzuki, /. Biochem., 89, 599 (1981). preparations by atomic absorption and 1,10- 13) E. B. Kearney, J. Biol. Chem., 235, 865 (1960). phenanthroline methods showed3 g atoms of 14) N. Mori, M. Sano, Y. Tani and H. Yamada, Agric. total iron permol of enzyme, and the ab- Biol. Chem., 44, 1391 (1980). 15) Y. Tani, N. Mori, K. Ogata and H. Yamada, Agric. sorption spectrum of the reduced enzyme in Biol. Chem., 43, 815 (1979). pyridine-NaOH solution showed maxima at 16) Y. Machida and T. Nakanishi, Agric. Biol. Chem., 413nm, 520nm, and 549nm (Fig. 6). These 48, 2463 (1984). results indicate that the enzyme contains a 17) M. Bruhmuller, H. Mohler and K. Decker, Eur. J. heme component. Inhibitions of the enzyme Biochem., 29, 143 (1972). activities by potassium cyanide and sodium