Purification and properties of d -amino-acid oxidase, an inducible flavoenzyme from Rhodotorula gracilis

136

Biochimica etBiophysicaActa 914 (1987) 136-142 Elsevier

BBA32895

Purification and properties of D-amino-acid oxidase, an inducible flavoenzyme

from R h o d o t o r u l a

gracilis

*

MireUa Pilone Simonetta, Maria A. Vanoni and Paola Casalin Department of General Physiology and Biochemistry, University of Milan, Milan (Italy) (Received29 January 1987) Key words: Yeast; D-Amino-acidoxidase; Flavoprotein; (R. gracilis) D-Amino-acid oxidase (D-amino-acid:oxygen oxidoroductase (deaminating), EC 1.4.3.3) was purified about 950-fold from the red yeast Rhodotorula gracilis. The procedure gave an enzyme preparation which is greater than 90% homogeneous on SDS-pulyacrylamide gels with a specific activity of 58 U / r a g at 3 7 ° C with D-alanine as substrate. D-Amino-acid oxidase from yeast is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalentiy, bound FAD. The subunit molecular weight is 37 000, while the native enzyme is a dimer of 79000 as determined by SDS-polyacrylamide gel electropboresis and gel-filtration chromatography, respectively. The enzyme from Rhodotorula oxidizes several D-amino acids and activity was also detected on thiazolidine-2-carboxylic acid. Substrate specificity and inhibition by benzoate differ from the ones exhibited by the mammalian enzyme and the recently identified D-amino-acid oxidase from Trigonopsis variabilis.

Introduction D-Amino-acid oxidase (D-amino-acid : oxygen oxidoreductase (deaminating); EC 1.4.3.3) catalyzes the oxidative deamination of D-amino acids, producing the corresponding a-keto acid and ammonia with concomitant reduction of molecular oxygen to hydrogen peroxide. Despite the fact that the presence of D-amino-acid oxidase has been reported in many organisms and that it is used as a marker of peroxisomes in higher organisms and microbodies in yeast [1,2], the only D-amino-acid oxidase species available in a homo* This paper is dedicated to the memoryof ProfessorMassimo Simonetta. Abbreviation: PMSF, phenylmethylsulfonylfluoride. Correspondence: M.P. Simonetta, Dipartimento di Fisiologiae Biochimica Generali, Universith degli Studi di Milano, 26, via Celoria, 20133 Milan, Italy.

geneous form and in large quantities so far is the one from pig kidney [3]. To contribute to our knowledge of the function and the structure of this very important enzyme, efforts were directed towards an alternative source among microorganism species. This approach was further encouraged by applications of D-amino-acid oxidase in both analytical and industrial work [4-8] for which the mammalian enzyme cannot be fully exploited. Up to now, the attempts to purify Damino-acid oxidase as a flavoprotein from microorganisms, particularly from yeast, have been hampered by the low concentration of the enzyme in the cells, its instability and the impossibility of using the well-established purification procedure of the D-amino-acid oxidase from pig kidney [9]. Our group has shown that D-amino-acid oxidase is constitutively present at a very low level in Rhodotorula cells, but its synthesis can be selectively induced by the presence of D-amino acids in the growth medium [5].

0167-4838/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)

137 We here describe the purification and properties exhibited by a preparation of D-amino-acid oxidase purified to near homogeneity from Rhodotorula gracilis cells and we give for the first time direct evidence that also yeast D-amino-acid oxidase is an FAD-containing flavoprotein oxidase. The purification of D-amino-acid oxidase from yeast will open up a variety of possibilities as far as the regulation and biogenesis of this enzyme are concerned, besides comparative studies with the mammalian enzyme. Materials and Methods

Materials DEAE-Sephacel, phenyl-Sepharose CL-4B, FPLC apparatus, ion-exchange Mono Q and gelfiltration Superose 12 columns were from Pharmacia Fine Chemicals (Uppsala, Sweden); Ultrogel AcA 34 was from LKB (Bromma, Sweden); cetylpyridinium bromide was from Fluka AG (Buchs, Switzerland). Marker proteins were from Pharmacia or Boehringer-Mannheim (Mannheim, F.R.G.). PMSF, pepstatin, snake venom phosphodiesterase and bovine heart catalase were from Sigma Chemicals (St. Louis, MO, U.S.A.). Iodonitrotetrazolium salt, yeast growth media and other reagents were all of analytical grade and purchased from Merck (Darmstadt, F.R.G.), OLThiazolidine-2-carboxylic acid was prepared as described in Ref. 10. Purified pig kidney D-aminoacid oxidase was a generous gift of Prof. B. Curti (Milan, Italy). Organ&m and cultivation R. gracilis cells (American Type Culture Collection, strain number 26217) were grown in an organic medium described elsewhere [5], supplemented with 15 mM DL-alanine at 30°C under stirring with aeration in a 14 litre New Brunswick Microferm Fermentor. After 34 h, cells were harvested by centrifugation and stored frozen. Enzyme assay and protein determination D-Amino-acid oxidase activity was assayed polarographically at 37°C with a Rank oxygen electrode. The standard assay mixture consisted of 75 mM sodium pyrophosphate buffer (pH 8.5), 56

mM DL-alanine and (when appropriate) 5 #M FAD. One unit of activity corresponds to the uptake of 1 /~mol of oxygen per min under our conditions. For determination of kinetic parameters and substrate specificity, assays were run under the same conditions, except for the substrate and its concentration. Protein concentration was determined using biuret [11], Lowry [12] or Bradford protein assays [13] according to the sample total protein content.

Purification of o-amino-acid oxidase The purification procedure was carried out at 0-4 ° C. Cell paste was resuspended (240 g/l) in 100 mM potassium phosphate (pH 7.5)/2 mM E D T A / 5 mM 2-mercaptoethanol/0.3% cetylpyridinium bromide/1 mM PMSF and homogenized with glass beads (0.5 mm diameter, 9 g / g yeast) in a refrigerated Vibrogen-Zellmtihle (Biihler, Tiibingen, F.R.G.) apparatus. The homogenate was centrifuged at 100000 x g for 30 min and the supernatant was precipitated between 30 and 60% saturation with ammonium sulphate. The enzyme pellet was resuspended in 10 mM potassium phosphate buffer (pH 7.5)/5 mM 2mercaptoethanol/2 mM EDTA/10% glycerol (buffer A). After dialysis against the same buffer the enzyme solution was applied to a 40.5 x 2.4 cm DEAE-Sephacel column and eluted with buffer A. Active fractions were combined, concentrated in an Amicon Ultrafiltration apparatus (YM 30 membrane) and loaded on a 93 x 1.5 cm Ultrogel AcA34 column. The column was eluted with 20 mM potassium phosphate buffer (pH 7.5)/5 mM 2-mercaptoethanol/2 mM EDTA/10% glycerol. Concentrated enzyme was adsorbed on a 9.3 x 1 cm phenyl-Sepharose CL-4B column. The enzyme was selectively eluted in the reduced form with buffer A containing 5 mM o-phenylglycine. The active fractions were concentrated and dialyzed against buffer A. The final purification step consisted of a FPLC gel filtration on a Superose 12 column equilibrated and eluted with 20 mM potassium phosphate buffer (pH 7.4)/0.25 M NaCl/0.3 mM EDTA/10% glycerol. Anion-exchange chromatography on FPLC Mono Q column could also be used as an alternative final step. The enzyme was loaded on the Mono Q

138 (

column and eluted in 20 mM Tris-HC1 (pH 7.85)/ 2 mM 2-mercaptoethanol/2 mM E D T A / 1 0 % glycerol. Pure enzyme was stored at - 8 0 ° C.

Electrophoretic methods and molecular-weight determination Disc-polyacrylamide gel electrophoresis was performed according to Davis [14]. Gels were stained for proteins with Coomassie blue R-250. Activity stain was obtained by incubating gels in 35 mM sodium pyrophosphate buffer (pH 8.5)/23 /~M F A D / 9 3 /~g/ml iodonitrotetrazolium chloride/65 mM DL-alanine at 37°C. The subunit molecular weight of D-amino-acid oxidase was determined by slab SDS-polyacrylamide gel electrophoresis following a modification of the method of O'Farrell [15]. A densitometric trace of gels was obtained with a LKB 2202 Ultroscan Laser Densitometer. Integration of the stains was used as an estimate of enzyme purity. The molecular weight of the native enzyme was also determined by gel filtration on a FPLC Superose 12 column equilibrated as previously described. Markers were run under the same conditions as the enzyme. Glycoprotein stain was carried out with the concanavalin A-peroxidase detection method of Hawkes [16], after blotting of samples from SDSpolyacrylamide gel electrophoresis on a nitrocellulose sheet. Flavin determination and identification Flavin concentration was determined spectrophotometrically using e455= 11.3 mM -1 • cm -1. The method of Forti and Sturani [17] was followed for the identification of flavin chromophore. Release of flavin was obtained by boiling purified enzyme for 3 min in the dark. Flavin fluorescence of the supernatant was analyzed before and after addition of excess phosphodiesterase. Absorption and fluorescence measurements were carried out in a Cary 219 spectrophotometer and a Jasco FP-550 spectrophotofluorometer, respectively. Results and Discussion

Previous attempts to purify yeast D-amino-acid oxidase were greatly frustrated by the poor extraction of the enzyme from cells and by its high

0.8

I

I

T

-0.16

0,6

UJ

0.4

0.08

o (It

0.2

i

300

400 [nm)

I

500

600

Fig. 1. Absorbance spectrum of a 0.476 mg/ml solution of D-amino-acid oxidase purified from Rhodotorula in 20 mM potassium phosphate buffer (pH 7.4)/0.25 M NaC1/0.3 mM EDTA/10% glycerol.

instability and susceptibility to proteolytic degradation. A detergent is required to properly solubilize yeast D-amino-acid oxidase. In our case, the presence of cetylpyridinium bromide in the homogenization medium greatly improved enzyme extraction from Rhodotorula cells and its stabilization during the first purification step. Glycerol was also found to stabilize the enzyme, and it was thus included in all buffers at a concentration of 10%; it must be noticed that the presence of substrate or benzoate was totally ineffective in stabilizing the protein. The stabilizing effect of cetylpyridinium bromide and glycerol suggests a high degree of hydrophobicity of solubilized protein. Table I shows the results of a representative purification procedure. In all chromatographic steps, enzyme activity was eluted as a single peak. The procedure yields a 950-fold purification factor with respect to the crude extract. The final enzyme preparation has a specific activity of 58 U / m g , which is about 3-fold higher than the one reported for D-amino-acid oxidase purified from Trigonopsis variabilis (21.7 U / m g ) [8] and the one we

139 TABLE I PURIFICATION OF D-AMINO-ACID OXIDASE FROM The activity was measured with DL-alanine as substrate. Starting material: 320 g of frozen cell paste. Step

Total activity (units)

Protein (mg)

1 Crude extract 2 30-60% (NH4)2SO 4 precipitate 3 DEAE-Sephacel chromatography 4 Ultrogel AcA 34 chromatography 5 Phenyl-Sepharose CL-4B chromatography 6 Superose 12 chromatography

1037

16 929

0.061

1

100

1101

4 042

0.272

4

106

800

224

3.6

59

77

570

57

10.0

164

55

303

6

50.5

828

29

162

2.8

57.8

948

16

measured for pig kidney enzyme (21.3 U / m g under standard assay conditions in the absence of added catalase, see also Ref. 9). The affinity chromatography step was particularly effective, since after this step the preparation shows a well-resolved visible absorption spectrum typical of a flavoprotein. The stability of the enzyme preparation is greatly dependent upon enzyme purity. The purified enzyme shows a good stability, since no change in electrophoretic patterns or significant loss of activity was detected within 2-3 months when stored frozen at - 8 0 o C. An alternative last step for enzyme purification using a FPLC Mono Q column can also be used instead of gel-filtration on Superose 12; however, some loss of FAD occurs. The protein stability shows a decrease at pH values higher than 8, and it is greatly influenced by temperature, since a sharp decrease in activity is observed above 35 o C. The absorption spectrum of D-amino-acid oxidase from Rhodotorula is shown in Fig. 1. The two maxima at 370 and 455 nm are typical of the flavin chromophore. E455 allows the calculation of 13 nmol flavin per mg of protein (determined by the Lowry method). The ratio E278/E455, which is frequently used to estimate the purity of a flavoprotein, is 12. This value is slightly higher than the one of about 9.5 determined for the mammalian enzyme [18]. The purified enzyme also shows a fluorescence emission maximum at 530 nm, as expected for a

Specific activity (U/rag)

Purification (fold)

Yield (%)

flavoprotein. Fluorimetric analysis of the extracted flavin in the presence of phosphodiesterase revealed its identity as FAD. Fig. 2 shows the

1

2

3

4

5

6

Fig. 2. Disc-polyacrylarnide gel electrophoresls of samples at different stages of purification. Gel was stained with Coomassic Blue. (1) ammonium sulphate precipitate; (2) after DEAESephacel chromatography; (3) after Ultrogel AcA 34 chromatography; (4) after phenyl-Sepharose chromatography, (5) after FPLC Superose 12; (6) activity staining of enzyme after FPLC Superose 12.

140

I

I

I

I

I

,

,

94000 _._67000 t--

_,,_43000

20100

14400

-1

2

2

~o

--51 U _9.0 o E

o~ o .J

•

3

Fig. 3. SDS-polyacrylamide gel electrophoresis of D-amino-acid oxidase after the two last alternative steps of purification. Samples (5 #g each) were incubated at 95°C for 12 min in 1% SDS and 2.5 mM 2-mercaptoethanol. (1) Mono Q chromatographed enzyme; (2) Superose 12 chromatographed enzyme; (3) marker proteins: phosphorylase B, bovine serum albumin, ovalbumin, soybean trypsin inhibitor, a-lactoalbumin; (marker molecular weights are shown in the figure).

pattern of samples at different stages of purification obtained by disc-polyacrylamide gel electrophoresis. In all cases only one protein band stains for D-amino-acid oxidase activity indicating both enzyme stability and lack of proteolytic degradation of the enzyme. Staining is strictly specific for D-amino-acid oxidase activity and it is not affected when FAD is omitted from the incubation mixture (not shown). Final D-amino-acid oxidase preparations obtained through either anion exchange or gel-filtration FPLC show two major protein bands, one of which stains for D-aminoacid oxidase activity. SDS-polyacrylamide gel electrophoresis of purified samples is shown in Fig. 3. The enzyme preparations obtained through either Mono Q or Superose 12 chromatography both migrate as a major protein band with an M r of 37000 _+ 1600 which represents more than 90% of total protein content of the sample. Traces of

104

,

,

I

Ve/vo

2

Fig. 4. Molecular weight of D-amino-acid oxidase (O) as determined by gel filtration on FPLC Superose 12. Marker proteins (t) were as follows (molecular weights are indicated in parentheses): ferritin (440000), catalase (240000), aldolase (158000), alkaline phosphatase (100000), bovine serum albumin (67000), ovalbumin (45000), chymotrypsinogen A (25000), cytochrome c (12500).

impurities in the electrophoretic pattern confirm the difficulties in obtaining homogeneous preparations. Staining for glycoproteins on nitrocellulose blots after SDS-polyacrylamide gel electrophoresis did not evidence any band, thus showing the absence of carbohydrate content in the enzyme (not shown). Molecular weight determination by gel-filtration of purified enzyme on a FPLC Superose 12 column gave a single symmetric peak with an M r of 79 000 ± 3500 (Fig. 4). Comparison of results obtained by gel-filtration, disc- and SDS-polyacrylamide gel electrophoresis suggests that the native protein is a dimer of identical subunits and that the inactive protein band present in disc-polyacrylamide gel electrophoresis is a form of the same D-amino-acid oxidase. On the basis of spectral data, subunit molecular

141 TABLE II SUBSTRATE SPECIFICITY OF OXIDASE F R O M R H O D O T O R U L A

D-AMINO-ACID

Activity measurements were carried out at both 20 and 40 mM D-amino-acid concentration as described in Materials and Methods. Activity is given in relation to the one measured on D-alanine taken as 100. Substrate

Concentration (mM)

D-Alanine o-Valine D-Leucine o-Isoleucine n-Proline D-Phenylalanine D-Phenylglycine D-Tryptophan D-Methionine D-Serine o-Threonine o-Cysteine o-Asparagine o-Glutamine o-Aspartic acid o-Glutamic acid o-Lysine o-Arginine o-Histidine DL-Thiazolidine-2carboxylic acid

20 40 20 40 40 20 40 20 20 20 40 40 40 20 20 20 20 20 20

a

Relative activity (%)

a. a a a a a

100 86 75 48 50 97 29 109 127 44 41 106 64 70 0 0 7 6 6

25 a

17

a

a a a a

a No significant difference was observed at higher substrate concentration.

weight and protein content determination a ratio of 0.5 mol FAD per mol monomer can be calculated. This value could be closer to 1 if the Lowry method overestimated protein concentration due to a high percentage of aromatic amino-acid contents, or if some apoenzyme was present in the final o-amino-acid oxidase preparation. In both cases a ratio E278/E455 higher than 9.5 would be justified. D-Amino-acid oxidase from Rhodotorula is a true oxidase, since it transfers electrons to molecular oxygen, yielding hydrogen peroxide. In fact, addition of excess catalase to the assay mixture halves oxygen consumption. At variance with pig kidney D-amino-acid oxidase, addition of excess FAD to the assay mixture does not increase the activity, either with the crude or with the purified enzyme. The enzyme shows a rather extended

interval of maximum activity between 37 and 50 ° C and a p H optimum between 8 and 9. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids. Moreover, the presence of the Lisomer does not interfere with D-amino-acid oxidase activity (not shown). Table II summarizes the substrate specificity of purified D-amino-acid oxidase. A large number of D-amino acids containing nonpolar or polar uncharged R groups are good substrates, whereas D-amino acids with negatively and positively charged groups are completely or partially ur/reactive. D-Methionine appears to be the best substrate together with Dphenylalanine, D-alanine, D-tryptophan and D-cysteine. Substrate specificity then differs markedly from that of the mammalian enzyme [19] where D-proline is by far the best substrate. This difference is confirmed by the low activity determined with DL-thiazolidine-2-carboxylic acid, the proposed physiological substrate of D-aminoacid oxidase from pig kidney enzyme [10,20]. The apparent K m calculated for this compound in our conditions is 2.3 mM, in comparison to a 0.27 mM value for the mammalian D-amino-acid oxidase at pH 8.3 and 23°C [20]. The enzyme is only slightly inhibited by benzoate, a substrate analogue and inhibitor of pig kidney D-amino-acid oxidase that binds at the enzyme active site [21,22]. A K i for benzoate of 1.04 mM has been determined for yeast D-amino-acid oxidase, i.e., about 3 orders of magnitude higher with respect to the one determined for the mammalian enzyme [22]. It must be noticed that the described procedure yields enzyme preparations with specific activities ranging from 20 to 60 U / m g , depending on the variability of the starting material. The E278/E45 ~ ratio reflects this variation and the electrophoretic patterns show the presence of some contaminants. However, in all cases, the visible absorption spectrum of the flavin is well resolved and the kinetic properties of the enzyme are identical. Furthermore, since in no case is an increase in activity observed when the enzyme is incubated with excess FAD, the presence of significant amounts of apoprotein can be ruled out. As for the well-characterized mammalian enzyme, D-amino-acid oxidase from Rhodotorula is a FAD containing oxidase with a subunit molecular

142 weight of a b o u t 40 000. However, our p r e l i m i n a r y data show some significant differences b e t w e e n the properties of the enzymes from the two sources, which make the yeast enzyme more suitable for biotechnological purposes. Finally, besides the possible application of this enzyme in analytical a n d industrial work, the purification of D-aminoacid oxidase from Rhodotorula, which leads to a highly purified a n d stable enzyme preparation, offers the possibility of a variety of comparative studies o n the characterization a n d regulation of this flavoprotein.

Acknowledgement This work was supported b y a grant from Ministero della P u b b l i c a Istruzione, R o m a , Italy.

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