Agric. Biol. Chem., 44 (6), 1383•`1389, 1980 1383

Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M-1

Nobuhiro MORI,* Bunsei KAWAKAMI,Yoshiki TANI and Hideaki YAMADA Departmentof AgriculturalChemistry, Kyoto University,Kyoto 606, Japan ReceivedFebruary 5, 1980

Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindro

carpon didymum M-1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method

and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption

spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 run. The

enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained

two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the

protein. The enzyme was inactivated by Ag+, Hg2+, Zn2+ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines.

Km and Vmax values for dimethylglycine were 9.1mM and 1.22ƒÊmol/min/mg, respectively.

The enzyme catalyzed the following reaction: Dimethylglycine+O2+H2O ?? sarcosine+form

aldehyde+H2O2.

It has been reported that dimethylglycine The present paper deals with the purifica and sarcosine were metabolized to sarcosine tion and some properties of dimethylglycine and glycine, respectively, by the oxidative oxidase from C. didymum M-1. demethylation reaction in liver mitochondria.1) Dimethylglycine dehydrogenase and sarco sine dehydrogenase have been partially purfied MATERIALS AND METHODS

from liver mitochondria of rat2,3) and Rhesus Materials. Hydroxyapatite was prepared accord monkey.4) On the microbial oxidation of ing to the method of Tiselius et al.9) Reference pro dimethylglycine, Shieh reported that dimethyl teins used for molecular weight determination by gel filtration, and pyruvate kinase, myokinase and lactate glycine was oxidized to sarcosine and formal dehydrogenase were purchased from Boehringer Man dehyde by intact cells of Achromobacter choli nheim GmbH. Phosphodiesterase of Crotalus ada

nophagum and sarcosine was further oxidized manteus venom was from Sigma Chemicals Co., Ltd. to glycine and formaldehyde by the cell ex Reference proteins used for molecular weight by SDS tract.5) disc gel electrophoresis were purchased from Pharmacia Fine Chemicals. We have previously reported that choline

was oxidized to betaine aldehyde by choline Microorganisms and cultivation. Cylindrocarpon oxidase in Cylindrocarpon didymum M-1.6) didymum M-1, which was able to grow on choline as the This enzyme catalyzed the first step of the cho sole carbon source, was used throughout this work. line oxidation. The enzyme was purified and The medium consisted of 1.0g of betaine, 0.1g of meat extract, 0.1g of KH2PO4, 0.1g of K2HPO4, 0.1g of characterized in details.7,8) Subsequently, we NaCl and 0.05g of MgSO4.7H2O in 100ml of tap attempted to purify the related three enzymes in water, pH 7.0. The cultivation was carried out at 28•Ž

choline metabolism by C. didymum M-1, be for 50hr under reciprocal shaking. taine aldehyde dehydrogenase, dimethyl Enzyme assay. Dimethylglycine oxidase activity glycine oxidase and sarcosine oxidase, and suc was assayed by the measurement of formaldehyde or ceeded to purify two enzymes, dimethylglycine H2O2 formed and by the measurement of oxygen con oxidase and sarcosine oxidase. sumed. The standard reaction system to estimate * Present address: Department of Agricultural formaldehyde formed contained 300ƒÊmol of Tris-HCl

Chemistry, Tottori University, Tottori 680. buffer (pH 9.0), 150ƒÊmol of dimethylglycine and a 1384 N. MORI, B. KAWAKAMI, Y. TANI and H. YAMADA

suitable amount of enzyme solution in a total volume RESULTS of 3.0ml. The reaction was carried out with shaking at 30•Ž for 10min. The reaction was stopped by the Purification of dimethylglycine oxidase addition of 0.1 ad of 4 N HCl. The activity to form All steps were performed at 0•`5•Ž. Tris

H2O2 was determined by the method coupled with HCl buffer (pH 8.5) containing 0,1mM dithio peroxidase, phenol and 4-aminoantipyrine. The reac theritol (DTT) was used and centrifugation tion mixture contained 300 pmol of Tris-HCl buffer was carried out at 12,000•~g for 20min through (pH 9.0), 6ƒÊmol of phenol, 4.5ƒÊmol of 4-aminoanti the purification procedure, unless otherwise pyrine, 6 unit of peroxidase, 150ƒÊmol of dimethyl glycine and a suitable amount of enzyme solution in a stated. total volume of 3.0ml. The reaction was started by the Step 1. Preparation of cell extract. addition of dimethylglycine, and the increase of absor bance at 500 nm was followed in a Hitachi double Washed cells (about 185g as wet weight) beam spectrophotometer. The oxygen consumption were suspended in 0.8 liter of 0.1M buffer and was measured with an oxygen electrode at 30•Ž. The disrupted for 50min with a Kaijo Denki reaction mixture contained 60ƒÊmol of Tris-HC1 buffer 19kHz ultrasonic oscillator. The cell extract (pH 9.0), 30ƒÊmol of dimethylglycine and a suitable was obtained by centrifugation. amount of enzyme solution in a total volume of 0.6 ml. One unit of dimethylglycine oxidase activity was de Step 2. Ammonium sulfate fractionation. fined as the amount of enzyme which catalyzed the for To 910ml of the cell extract was added 207g mation of 1ƒÊmol of formaldehyde or H2O2 per min and of solid ammonium sulfate to 30% saturation the consumption of 1ƒÊmol of oxygen per min. Speci fic activity was defined as the unit per mg of protein. under stirring, by adjusting pH to 8.5 with 14% ammonium hydroxide solution. After stirring Analytical method. Formaldehyde was determined for 30min, the precipitate formed was re according to the method of Nash.10) Sarcosine was moved by centrifugation and discarded. The determined by an automatic amino acid analyzer. Protein concentration for dimethylglycine oxidase was ammonium sulfate concentration of the super

determined by the absorbance at 280 nm, where natant solution was increased 50% saturation. E1%1cm value of 15.0 was used after dry weight determina The precipitate formed was collected by centri

tion. AMP was estimated by an enzymatic method fugation and was dissolved in 10mm buffer. with myokinase.11) The solution was dialyzed against four changes

Molecular weight determination. The molecular of the same buffer.

weight was determined by the method of Andrews. 1V) Step 3. DEAE-cellulose column chromato Sephadex G-200 was packed to a column (0.9•~100cm) and equilibrated with 50mm of Tris-HCI buffer (pH graphy. The dialyzed solution was placed

8.5) containing 0.1M NaCl and 0.1mm dithiothreitol. on a DEAE-cellulose column (5•~50cm)

The flow rate was 1ml/hr and each 0.5ml fraction was equilibrated with 10mM buffer. After wash collected. Cytochrome c, chymotrypsinogen A, egg ing the column with 70mM buffer, the enzyme albumin, bovine serum albumin, aldolase, catalase and was eluted with 70mM buffer containing 0.2M ferritin were used as the reference protein. NaCI. Active fractions were pooled and con

Disc gel electrophoresis. Electrophoresis in acryl centrated by the addition of ammonium sul amide gel was performed at a current of 2.0 mA per fate to 70% saturation. The precipitate ob gel in Tris-glycine buffer (pH 8.3) according to the me tained by centrifugation was dissolved in a thod of Davis.13) SDS-disc gel electrophoresis was minimal volume of 10mM buffer and dialyzed performed according to the method of Weber and Osborn.l4) Dimethylglycine oxidase was incubated in against the same buffer.

10mM of sodium phosphate buffer (pH 7.0) containing Step 4. Ammonium sulfate fractionation. 1% sodium dodecylsulfate and 2% 2-mercaptoethanol

for 3 hr at 60•Ž before the electrophoretic run. To the dialyzed enzyme solution solid am monium sulfate was added to 30% saturation. Ultracentrifugal analysis. The sedimentation velo After stirring for 30min, the precipitate formed city was measured with a Spinco model E analytical was removed by centrifugation. The am ultracentrifuge at 20•Ž and 59,700 rpm. Diffusion

constant was measured with the same apparatus operat monium sulfate concentration was then in

ing 12,590 rpm with the synthetic boundary cell. creased to 45% saturation. The precipitate Dimethylglycine Oxidase from Cylindrocarpon didymum M-1 1385

formed was collected by centrifugation and Step 7. Gel filtration by Sephadex G-150. dissolved in a minimal volume of lo mm The enzyme solution was placed on a Sephadex buffer and dialyzed against the same buffer. G-150 column (2.5•~120cm) equilibrated

with 50mM buffer containing 0.1m NaCI . Step 5. DEAE-Sephadex A-50 column The enzyme was eluted with the same buffer . chromatography. The dialyzed enzyme was The elution pattern of the enzyme is shown in placed on a DEAE-Sephadex A-50 column Fig. 2. The purification procedure is sum (3•~45cm) equilibrated with 0.1M buffer and marized in Table I. the column was washed with the same buffer.

The enzyme was eluted with an increasing linear gradient of NaCI concentration from 0 to 0.2M (Fig. 1). Active fractions were combined and concentrated by the addition of ammonium sulfate to 70% saturation. The precipitate formed was collected by centrifugation and was dissolved in 10mM potassium phosphate buffer containing 0.1 mm DTT.

FIG. 2. Elution Pattern of Sephadex G-150 Column Chromatography.

The flow rate was 15ml per hr and 3ml fractions were collected. (•›), absorbance at 280nm; (•œ), enzyme

activity.

TABLE 1. SUMMARY OF PURIFICATION OF FIG. 1. Elution Pattern of DEAE-Sephadex A-50 DIMETHYLGLYCINE OXIDASE FROM Column Chromatography. Cylindrocarpon didymunr M-1 Fractions of 5ml were collected. (•›), absorbance at 280nm; (•œ), enzyme activity.

Step 6. Hydroxyapatite column chromato graphy. The dialyzed enzyme solution was placed on a hydroxyapatite column (2.5 20cm) equilibrated with 10mM potassium•~ phosphate buffer (pH 8.0) containing 0.1mM DTT. The column was washed with 70mM potassium phosphate buffer (pH 8.0) containing 0.1mM DTT. The enzyme was eluted with 70mM potassium phosphate buffer containing

0.3 M ammonium sulfate and 0.1mM DTT.

Active fractions were combined and con centrated by the addition of ammonium sulfate to 70% saturation. The precipitate formed Homogeneity was collected by centrifugation and dissolved The purified enzyme sedimented as a single in a minimal volume of 50mM buffer containing and symmetric peak in the ultracentrifuge in 0.1M NaCl. 0.1M potassium phosphate buffer, pH 8.0 (Fig. 1386 N. MORI, B. KAWAKAMI, Y. TANI and H. YAMADA

FIG. 3. Sedimentation Patterns of Purified Enzyme. Protein concentration was approximately 0.44% in 0.1M phosphate buffer (pH 8.0).

from 2.4 to 4.4mg per ml of 0.1M potassium

phosphate buffer (pH 8.0) and the partially specific volume of the enzyme was assumed

to be 0.74. The diffusion coefficient in water at 20•Ž was 3.95•~10-7cm2/sec. From these

values, the molecular weight of the enzyme

was calculated to be about 180,000 according

to the equation of Svedberg and Pederson.1 )

On the other hand, the molecular weight was estimated to be 170,000 by gel filtration on

Sephadex G-200 (Fig. 5).

Subunit composition of the enzyme was determined by disc gel electrophoresis analysis

of the enzyme in the presence of 1% sodium

dodecylsulfate and 2% 2-mercaptoethanol.

Molecular weight of subunit of the enzyme was estimated to be 82,000 (Fig. 6). These

results showed that dimethylglycine oxidase

might be composed of two identical subunits.

FIG. 4. Polyacrylamide Disc Gel Electrophoresis of

Purified Enzyme.

(A), Purified enzyme (18ƒÊg protein) was applied to electrophoresis in the absence of SDS with 7.5% gel.

The gel was stained with 1% amino black in 7 acetic acid. (B), Purified enzyme (3.5ƒÊg protein)

was applied to electrophoresis in the presence of SDS

with 7.5% gel. The gel was stained with Coomassie

Brilliant Blue.

3). The enzyme gave a single band on poly

acrylamide and SDS-polyacrylamide gel ele FIG. 5. Determination of Molecular Weight of the ctrophoresis, respectively (Fig. 4). Enzyme by Gel Filtration. Experimental conditions are described in MATERIALS

Physicochemical properties of enzyme AND METHODS. A: Cytochrome c (M. W. 12,500), B: Chymotrypsinogen A (M. W. 25,000), C: Egg al Molecular weight. The sedimentation bumin (M. W. 45,000), D: Aldolase (M. W. 158,000), coefficient in water at 20•Ž (S2020,w) was 7.6 S, E: Dimethylglycine oxidase, F: Catalase (M. W. where the protein concentration was varied 240,000), G: Ferritin (M. W. 450,000). Dimethylglycine Oxidase from Cylindrocarpon didymurn M-l 1387

was not identical with FAD, FMN and riboflavin on a thin layer plate. These results evidenced the presence of a covalently bound flavin in the enzyme. In order to discriminate between FMN and FAD an attempt was made to determine AMP after treatment of the purified enzyme with pronase and venom phosphodiesterase. After heat inactivation, AMP was found in the supernatant to be 2 mol FIG. 6. Determination of Molecular Weight of the per mol enzyme (Table II). Therefore, the Enzymeby SDS-discGel Electrophoresis. enzyme was determined to contain FAD, Experimentalconditions are describedin MATERIALSwhich was covalently bound to the enzyme ANDMETHODS. A: Phosphorylaseb (M. W. 94,000), protein. B: Dimethylglycineoxidase, C: Bovineserum albumin

(M. W. 68,000),D: Egg albumin (M. W. 43,000),E: TABLE II. IDENTIFICATION OF FAD BY Carbonicanhydrase (M. W. 30,000). LIBERATION OF AMP

Dimethylglycine oxidase (27.1 nmol) and FAD

Identification of prosthetic group. Absorp (123 nmol) each in 1.4ml of 50mM Tris-HC1 buffer tion spectrum of dimethylglycine oxidase (pH 8.5) were incubated with 4 mg of pronase at 37•Ž showed maxima at 277, 345 and 450nm for 16 hr. The heat-inactivated (3 min) digest was lyophilized and resolved in 0.4ml of water. The (Fig. 7). Extinction coefficient of the enzyme solution was treated with 50ƒÊg of phosphodiesterase at 280 nm (E1%) 1cm and ratio of E280nm/E450nm for 30min. The reaction was stopped by heating and was 15.0 and 22.2, respectively. Addition of AMP was assayed enzymatically in 0.3ml aliquot of dimethylglycine to the enzyme under anaerobic the enzyme solution and 0.2ml aliquot of FAD solu conditions resulted in the disappearance of the tion, respectively. peak at 450 rim. Treatment of the enzyme in 5% trichloroacetic acid for 10min in a boil ing water bath did not show any release of colored material in the soluble fraction. Tryptic-chymotryptic digest of the enzyme

Some enzymatic properties

The enzyme was most active at pH 8.5 to

9.0 (Fig. 8) and stable at pH between 6.0 and 7.5, when incubated at 40•Ž for 15min. The

enzyme was active toward only dimethylgly

cine but completely inert toward choline, be

taine, sarcosine, creatine, creatinine, carni

tine, N, N-dimethylaminoethanol and alkyl

amines. Kinetic parameters, Kin and Vmax

for dimethylglycine were estimated by Linewe aver-Burk plots. Km value of 9.1mM and

Vmax value of 1.22ƒÊmol/min/mg for dimethyl

FIG. 7. Absorption Spectra of the Enzyme and the glycine were obtained. Reduced Enzyme. The stoichiometry of oxygen uptake and Curve A: The native enzyme solution at a concentra sarcosine and formaldehyde and hydrogen tion of 4.1mg/ml in 0.05M Tris-HCl buffer (pH 8.5) peroxide formation during dimethylglycine containing 0.1mM DTT. oxidation was examined. An aliquot of the Curve B: The reduced enzyme after the addition of

6ƒÊmol of dimethylglycine under anaerobic conditions. reaction mixture was removed, and sarcosine, 1388 N. MORI, B. KAWAKAMI, Y. TANI and H. YAMADA

TABLE III. STOICHIOMETRY OF THE

DIMETHYLGLYCINE OXIDATION

The purified enzyme of 60ƒÊg of protein was used.

The reaction was carried out in a Gilson respirometer

for 10min under the standard assay conditions.

formaldehyde and H2O2 were estimated by amino acid analyzer, Nash's and 4-amino phenazone method, respectively. As shown in Table III, it was found that each 1mol of FIG. 8. Effect of pH on the Enzyme Activity. sarcosine, formaldehyde and H2O2 was pro Enzyme activity was assayed by the determination of duced per mol of oxygen consumed. oxygen consumed under the standard assay conditions Effect of metal ions and some chemicals with 0.1M buffer as indicated. Tris-maleate buffer was used from pH 6.5 to 8.5, Tris-HCI buffer from pH on activity of the enzyme are summarized in 7.5 to 9.0, and ammonium buffer from pH 8.0 to 10.5. Table IV. Ag+, Hg+, Zn+ and iodoacetate inhibited the enzyme activity but p-chloromer TABLE IV. EFFECT OF METAL IONS AND curibenzoate not inhibited the activity at the CHEMICALS ON DIMETHYLGLYCINE OXDASE ACTIVITY concentration of 0.1mM. The enzyme activity was assayed by the determina tion of oxygen consumed under the standard condi DISCUSSION tions. The enzyme was preincubated for 10min with the metal ions or chemicals before the addition of The enzymatic conversion of dimethylglycine substrate. and sarcosine to formaldehyde was first ob served in 1941 by Handler et al. in rat liver homogenate.16) Mackenzie et al. reported that dimethylglycine and sarcosine were metabolized to sarcosine and glycine, respec tively, to form formaldehyde by phosphate washed mitochondria from liver.1) Dimethyl glycine dehydrogenase and sarcosine dehydro genase were solubilized from mitochondria of rat liver by sonic irradiation, and then purified and characterized.2,3) Both enzyme were found to require an electron transfer flavopro tein in order to reduce 2, 6-dichlorophenolin dephenol.17) The electron transfer protein can be replaced by phenazine methosulfate. On microbial oxidation of dimethylglycine to sarcosine, Shieh briefly reported that dimethyl glycine was oxidized to sarcosine to form for maldehyde by intact cell of Achromobacter cholinophagum.5) But, the detail of the reac * p-Chloromercuribenzoate tion has not been clarified enzymatically. ** 5, 5'-Dithio-bis (2-nitrobenzoic acid). Dimethylglycine oxidase purified and chara Dimethylglycine Oxidase from Cylindrocarpon didymum M-1 1389 cterized in this work was found to be a new REFERENCES type of enzyme which oxidized dimethylglycine 1) C. G. Mackenzie and W. R. Frisell, J. Biol. Chem., to sarcosine. The enzyme catalyzed oxidative 232, 417 (1958). demethylation of dimethylglycine to form of 2) D. D. Hoskins and C. G. Mackenzie , ibid., 236, sarcosine, formaldehyde and hydrogen pero 177 (1961). xide. 3) W. R. Frisell and C. G. Mackenzie, ibid., 237, The purified enzyme showed an absorption 94 (1962). 4) D. D. Hoskins and R. A. Bjur, ibid., 239, 1856 spectrum characteristic of a flavoprotein. The (1964). flavin moiety did not dissociate from the 5) H. S. Shieh, Can. J. Microbiol., 11, 375 (1965). enzyme protein by the treatment of boiling or 6) Y. Tani, N. Mori and K. Ogata, Agric. Biol. acid extract. After treatment by pronase and Chem., 41, 1101 (1977). 7) Y. Tani, N. Mori, K. Ogata and H. Yamada, ibid., phosphodiesterase, prosthetic group of the 43, 815 (1979). enzyme was identified FAD which was con 8) H. Yamada, N. Mori and Y. Tani, ibid., 43, 2173 tained two mol per mol of the enzyme. How (1979). ever, the binding form between flavin moeity 9) A. Tiselius, S. Hjerton and O. Levin, Arch. Bio and enzyme protein is unknown. We purified chem. Biophys., 65, 132 (1956). three kinds of oxidase, choline oxidase,7,8) 10) T. Nash, Biochem. J., 55, 416 (1953). 11) D. Jaworek, W. Gruber and H. V. Bergmeryer, dimethylglycine oxidase and sarcosine oxidase "Method of Enzymatic Analysis ," Academic from C. didymum M-1 to homogeneous state. Press, New York, 1974, pp. 2051-2055, All of these enzymes contained covalently 12) P. Andrews, Biochem. J., 96, 595 (1965). bound FAD as prosthetic group. These 13) B. J. Davis, Ann. N. Y. Acad. Sci.,121, 404 (1964). results are of interest in connection with the 14) K. Weber and M. Osborn, J. Biol. Chem., 244, biosynthesis of covalently bound flavin. 4406 (1969). 15) T. Svedberg and K. O. Pederson, "Ultracentri Acknowledgment. We are indebted to Dr. Riki fuge," Oxford University Press, 1940. maru Hayashi, Associate Professor of Kyoto Univer 16) P. Handler, M. L. C. Bernheim and J. R. Klein, sity, for taking the amino acid analysis. We wish to J. Biol. Chem., 138, 211 (1941). thank Professor Yoshio Ichikawa, Tottori University, 17) W. R. Frisell, J. R. Cronin and C. G. Mackenzie, for his encouragement during the course of this study. ibid., 237, 2975 (1962).