Agr. Biol. Chem., 40 (10), 2077•`2084, 1976

Characterization of Choline Dehydrogenase from Pseudomonas aeruginosa A-16

Toru NAGASAWA,Nobuhiro MORI, Yoshiki TANI and Koichi OGATA Departmentof AgriculturalChemistry, Kyoto University,Kyoto ReceivedJune 11, 1976

A choline dehydrogenase, which was present in the particulate fraction of the cell-free

extract of Pseudomonas aeruginosa A-16, oxidized choline to betaine aldehyde without any dissociable coenzymes, while the enzyme, which was treated with Triton X-100, oxidized

choline only with a supplement of phenazine methosulfate. The difference spectrum showed

the presence of cytochrome-like components in the particulate. Km values for choline and

phenazine methosulfate were 1.7•~10-3M and 1.4•~10-4M, respectively. The dehydrogenase was inhibited by SH-reagents such as p-chloromercuribenzoate and iodoacetic acid. Of a

variety of substrates tested, only choline caused the enzymatic reduction of phenazine metho

sulfate. The estimation of choline was tried using the enzyme.

It has been known that choline is metabo ƒÊ moles of potassium phosphate buffer (pH 7.4), 3.0 lized to betaine via betaine aldehyde by choline ƒÊ moles of potassium cyanide, 0.29 ƒÊmole of DCPIP dehydrogenase (EC 1.1.99.1) and betaine and an appropriate amount of enzyme. Standard assay of enzyme activity was performed with 0.7 ƒÊmole aldehyde dehydrogenase (EC 1.2.1.8) in mito of PMS and the assay was started by the addition of

chondria of mammalian liver. Since the 100 ƒÊmoles of choline chloride. The decrease in ab initial study of Bernheim and Bernheim,1„ sorbance at 600nm was followed. The enzyme assay ,2) the oxidation of choline has been studied only was performed at 30°C in a Hitachi double beam with a mammalian liver. spectrophotometer model 124. The extinction co Recently, we showed that the bacterial efficient for oxidized DCPIP at 600 nm was taken to be 21.5•~l03 liter-mol-1•.cm-1.4) The initial reaction choline dehydrogenase was present in the velocity was proportional to enzyme concentration up particulate fraction of the cell-free extracts of to a velocity of at least 0.18 ƒÊmole of DCPIP/min and Pseudomonas aeruginosa A-16.3) In this paper, the progress curve was linear with time for the first the characterization of the bacterial choline minute throughout this range. The unit of enzyme dehydrogenase will be described. activity is defined as being the amount of enzyme that catalyzes the reduction of 1 ƒÊmole of DCPIP/min under the standard assay conditions. Specific ac

MATERIALS AND METHODS tivity was defined as the units per mg of protein.

Materials. 3-(4,5-Dimethylthiazolyl-2)-2,5-di Protein determination. Protein was determined by phenyltetrazolium bromide (MTT) was purchased from the procedure of Lowry et al.,3) or from the absorbance Sigma Co. NAD, NADP and yeast extract were ob at 280 nm with the assumption of E0.1 1cm=1.0. tained from the Oriental Yeast CO., Cytochrome c (horse heart) was purchased from the Boehringer Co., Partial purification of the enzyme. The following Mannheim. Peptone, meat extract and casamino acid buffer mixtures were used: A, 0.01M potassium were obtained from Daigo Co., Ltd., Mikuni Co., Ltd. phosphate buffer (pH 7.4) containing 0.001% 2- and Difco Laboratory, respectively. All other com mercaptoethanol and 1mM EDTA; B, buffer A con pounds were of reagent grade and from commercial taining 10% glycerol. All procedures were performed sources. at 0•`5•Ž. Unless otherwise stated, centrifugation was carried out for 20 min at 12,000xg. Choline dehydrogenase assay. Phenazine metho sulfate (PMS)-2,6-dichlorophenol-indophenol (DCPIP) 1. Preparation of the particulate enzyme. The

assay system was used in this study, unless otherwise particulate enzyme was prepared as described in a stated. The assay medium (2.74ml) contained 125 previous paper.3) The specific activity was 5.73x 2078 T. NAGASAWA, N. MORI, Y. TANI and K. OGATA

10-3, and it was stored at -20•Ž for several months RESULTS without loss of activity.

Identification of reduction product 2. Triton treatment. To the preparation from 1,

Triton X-100 was added to a final concentration of The reaction mixture (20ml) containing 0.3%. with stirring. After the incubation for 30 min 100mg of choline chloride, 200ƒÊmoles of at 25•Ž, the solution was centrifuged at 100,000xg for potassium phosphate buffer (pH 7.4) and 2 60 min and gelatious precipitates were removed. The units of particulate enzyme was incubated for choline dehydrogenase almost appeared in the red and 6hr at 25•Ž with constant shaking. The deri transparent supernatant solution (specific activity: vative of the reaction product prepared ac 6.86•~10-3). The supernatant was dialyzed against the buffer B for 15 hr. cording to Jellinek's method6) was dissolved in

ethanol and crystallized by the addition of 3. Chloroform treatment. To the dialysate from water. After three recrystallization, the. pro 2, precooled chloroform was added with vigorous duct showed the melting point of 178•`180•Ž stirring to a final concentration of 33%. The tubid

solution was stirred for 20min, and centrifuged at (lit. mp 181•`183•Ž).6) The formula, 6,000xg. The aqueous solution was dialyzed for 15 hr C17H17011N8 was calculated from the analytical

against buffer B, and then concentrated by Sephadex results (calcd: C 40.00, H 3.53, Picric acid G-50 and ultrafiltration (specific activity: 14.6•~ 10-3). 44.7; found: C 39.80, H 3.62, Picric acid

45.00). Thus, the reaction product was 4. Sepharose 6B column chromatography. The

concentrated enzyme solution (2.0ml) was placed on a identified as the picrate of 2,4-dinitrophenyl-

Sepharose 6B (2.5•~73cm) column which had been hydrazone of betaine aldehyde. equilibrated with 0.01M buffer B containing 0.1M

NaCI and eluted with the same buffer at a flow rate of Electron carrier requirements for choline oxida 13ml/hr. Active fractions with a specific activity of 14.6•~10-3 were combined and concentrated by Sepha- tion

dex G-50 and ultrafiltration. The enzyme solution was As shown in Fig. 2, the particulate enzyme rechromatographed on the same column under the formed betaine aldehyde without requiring same condition. A typical elution profile is shown in any dissociable coenzymes. The addition of Fig. 1. After active fractions above specific activity of 14.9 were combined and concentrated, the enzyme potassium cyanide thoroughly inhibited the

solution (specific activity: 15.1•~10-3) was used as the formation of betaine aldehyde at the final

partially purified enzyme in this study. concentration of 1 mm. In contrast, Triton treated enzyme did not catalyze the choline

oxidation. The enzyme exhibited its catalytic

activity by an addition of PMS. The choline oxidation in the presence of PMS was insensi

tive to potassium cyanide. The relative ac

tivities with several electron acceptors in the

particulate enzyme or the purified enzyme were compared (Table I). The reaction of the

particulate enzyme with ferricyanide was con siderably significant, and the reaction with

externally added DCPIP, MTT or cytochrome

c measured only a part of the activity, and

that with NAD and NADP is almost negligible.

The reactions of Triton-treated enzyme with additions of ferricyanide, DCPIP , MTT and cytochrome c were completely abolished.

FIG. 1. Elution Diagram of the Fractionation of The reactivity of choline dehydrogenase with

Bacterial Choline Dehydrogenase on Sepharose 6B . PMS was fully retained under these condi

•œ, absorbance at 280 nm; •›, enzyme activity . tions. Bacterial Choline Dehydrogenase 2079

TABLE I. RELATIVE RATES OF CHOLINE OXIDATION WITH SEVERAL ELECTRON ACCEPTORS

The specific activities of particulate enzyme and Triton-treated enzyme used in this experiment were 7.76•~10-3 and 14.5•~10-3, respectively, in the stand ard assay of PMS-DCPIP system. All measurements

were based on initial rate and were calculated as moles of electron acceptors reduced per min per mg of protein. Rates were Vmax. PMS assay was car ried out under the standard condition. Ferricyanide assay was carried out by following the decrease in absorbance at 420nm. To the mixture (2.7ml) con taining 125 ƒÊmoles of potassium phosphate buffer FIG. 2. Time Course of Betaine Aldehyde Forma (pH 7.4), 7.9 ƒÊmoles of potassium ferricyanide and tion by Choline Dehydrogenase. 100 ƒÊmoles of choline chloride, an appropriate amount

The formation of betaine aldehyde was measured as of enzyme was added. The extinction coefficient for its 2,4-dinitrophenylhydrazone according to Jellinek's oxidized ferricyanide at 420nm was taken to be method.8) Reactions were catalyzed by 6.28mg of 1.02•~103 liter-mol-1. cm-1.7) DCPIP, MTT, cyto chrome c and NAD(P) as primary electron acceptor particulate enzyme (specific activity: 5.73•~10-3) or 4.15mg of Triton-treated enzyme (specific activity: were tested in the assay medium (2.74ml) containing 6.87•~10-3) in the reaction mixture (5.0ml) contain 125 ƒÊmoles of potassium phosphate buffer (pH 7.4), ing 208 ƒÊmoles of potassium phosphate buffer (pH 3.0 ƒÊmoles of potassium cyanide, 0.29•`1 ƒÊmole of 7.4), 189 ƒÊmoles of choline chloride. Phenazine primary electron acceptor, and an appropriate amount methosulfate and potassium cyanide were added to of enzyme. The assays were started by the addition the reaction mixture at the concentration of 0.42mM of 100 ƒÊmoles of choline chloride. Reaction rates and 1mM, respectively.•›•\•› were assayed at 600,550 and 340nm, respectively.7,8) , (A) particulate choline dehydrogenase. (B) particulate choline dehydrogenase+potassium cyanide. •œ•\•œ , (C) Triton-treated choline dehydrogenase. (D) Triton-treated choline dehydrogenase+phenazine methosulfate.

Reduction of cytochrome in particulate by

choline

The difference spectrum of particulate en zyme between the presence and the absence of choline showed the absorption maxima at

559nm, 520•`528nm and 427nm, which CDH* : choline dehydrogenase activity. seemed to be linked to the respiratory chain

FIG. 3. Difference Spectrum of Particulate Choline Dehydrogenase.

Two cuvettes contained 0.83 unit of particulate enzyme (protein 36.6mg), 125 ƒÊmoles of potassium

phosphate buffer (pH 7.4) and 1 ƒÊmole of potassium cyanide in 2.53ml of total volume. Choline chloride (100 ƒÊmoles dissolved in 0.1ml) was added to the sample cuvette, and 0.1ml of water to the

reference cuvette. 2080 T. NAGASAWA, N. MORI, Y. TANI and K. OGATA

such as cytochrome system (Fig. 3). Addition measured by heating at various temperature of sodium dithionite at the final concentration for 10 min in 0.1 M Tris-HCL buffer (pH 8.3). of 1mM intensified the peaks at 550nm, The loss of the initial activity was 10,50 and

520nm, 470nm and 421nm. The presence of 100% at 35,40 and 50•Ž, respectively. When

some kinds of cytochromes in this fraction the enzyme was kept at 37•Ž for 10min at could be conceivable. various pH values, the enzyme was stable

between pH 8.0 and pH 9.5, and 50% and

Properties of partially purified enzyme 25% of the initial activity was lost at pH 10.5

Several properties of the choline dehydro and pH 6.5 or pH 11.0 and pH 6.0, respectively.

genase were investigated with the partially 2) Effect of pH on the activity. The pH purified enzyme, which still contained about optimum was around pH 9.0, as shown in 20% impurity as the higher sedimentation Fig. 5. Tris-HCL buffer showed inhibitory component in an ultracentrifuge. effect on the enzyme.

1) Stability. Though the enzyme was 3) Substrate specificity. Various alcoholic stable to the storage at -20•Ž, the complete compounds at the concentration of 6mM was loss of the activity in the partially purified examined as the substrate in the standard enzyme was observed with the storage at 5•Ž assay mixture containing 0.3 unit of enzyme. for 5 or 6 days (pH 7.4). The addition of glyce The enzyme showed the high specificity against rol at the final concentration of 30 % promi choline. Following compounds were inactive: nently stabilized the enzyme activity (Fig. 4). 2-dimethylaminoethanol, 3-dimethylamino-l- The partially purified enzyme was more stable propanol, 1-dimethylamino-2-propanol, 2- than the particulate enzyme. amino- l -butanol, 2-amino- 2 -methyl- l -pro The thermal stability of the enzyme was panol, 2-(2-aminoethyl-amino)ethanol, mono ethanolamine, isopropanolamine, triethanol

amine, trimethylene glycol, methanol, isopro-

FIG. 4. Effect of Glycerol on the Stability of Choline FIG. 5. Effect of pH on Choline Dehydrogenase. Dehydrogenase. Following buffers were used: Potassium phosphate Two kinds of preparation of choline dehydrogenase buffer (•›•\•›) for pH 6 to 8; Tris-HCI buffer(•œ•\•œ) were dialyzed against 0.01M Tris-HCL buffer, pH 8.3, for pH 8 to 9; Glycine-HC1 buffer (•›•\•›) for pH 9 to containing 1mM of EDTA, 0.001% of 2-mercapto 11. The reaction mixture (2.65ml) contained 100 ethanol with or without 30% of glycerol for 15hr ƒÊ moles of choline chloride, 1.1 ƒÊmoles of PMS, 110 , and then the dialysates were stored at 5•Ž to measure ƒÊ moles of various kinds of buffers and 0.024 unit of enzyme. The reaction was carried out at 30•Ž and the remaining activity at 2•`3 days intervals . was stopped by soaking in boiling water. The amount •œ, particulate enzyme; •›, partially purified enzyme. of 2,4-dinitrophenylhydrazone of betaine aldehyde (A) glycerol (0%), (B) glycerol (30%). was measured at 440nm by Tellinek's method6) Bacterial Choline Dehydrogenase 2081

TABLE II. EFFECT OF METAL IONS AND VARIOUS panol, tert-butanol, isobutanol, n-amylalcohol, isoamylalcohol, tert-amylalcohol, anisalcohol, REAGENTS ON CHOLINE DEHYDROGENASE

cinnamylalcohol and ƒÀ-phenylethylalcohol. The reaction was carried out under the standard condition. Each inhibitor was added at the final The other miscellaneous compounds contain concentration as indicated. ing hydroxyl group such as DL-carnitine,

DL-threonine, DL-homoserine, tropine and

panthenol were also not attacked by the enzyme. The apparent Michaelis constant estimated

from double reciprocal plots was 1.7•~10-3M

for choline at pH 7.4 and 30•Ž. The apparent Michaelis constant for PMS was determined

to be 1.4•~10-4M in the presence of 3.4mM of choline.

4) Effect of metal ions and various reagents.

Effect of various metal ions and other com

pounds on the enzyme were examined (Table II). AgNO3, CuSO4, MnCl2, and HgCl2, caused

100% inhibition at the concentration of 1mM, p-CMB* : p-chloromercuribenzoate.

and an appreciable loss was observed with TABLE III. EFFECT OF VARIOUS COMPOUNDS ON SH-reagents such as p-chloromercuribenzoate, CHOLINE DEHYDROGENASE mercuric acetate and iodoacetic acid. CaCl2, The reaction was carried out under the standard SrCl2, MgCl2 and FeCl3 had little or no effect condition. Each compound was added at the final at the concentration of 1mM. Chelating concentration of 1mM. reagents, such as EDTA, o-phenanthroline, ƒ¿

,ƒ¿'-dipyridyl, sodium cyanide and sodium azide, did not cause the inhibition of the

enzyme activity at the concentration of 1mM.

5) Effect of choline analogues on the ac tivity. A variety of compounds bearing resemblance to substrate or product were

tested as an inhibitor of the enzyme (Table III). All of them did not exhibit the prominent

effect, though more or less inhibition was

recognized.

Application of choline dehydrogenase to choline estimation Choline which was recrystallized three times

From the above experiments, it seemed that from methanol were incubated with excess of choline dehydrogenase of P. aeruginosa A-16 enzyme (0.86 unit), PMS (0.7ƒÊmole), MTT

might serve as a specific analytical tool for the (0.43 ƒÊmole) and potassium phosphate buffer quantitative estimation of choline based on the (pH 7.4) (1.3 mmoles) in a total volume of reduction of MTT coupled with PMS . As the 2.88ml for 15min at 30•Ž. The formation of absorbance of MTT at 570nm increased with reduced MTT was linear when 5 ƒÊg to 15ƒÊg the reduction in contrast to DCPIP , PMS of choline was used under the standard condi - MTT system was more suitable for the estima tion (Fig. 6). The amount of reduced MTT

tion of choline than PMS-DCPIP system. was approximately 100% of the theoretically 2082 T. NAGASAWA, N. MORI, Y. TANI and K. OGATA

ured by the assay method. Therefore this experiment was carried out again by using PMS-DCPIP assay (Table V). Strong choline dehydrogenase activity was found in acetyl. choline-, choline-, and betaine-grown cells. No activity was detected in the cells grown on glucose medium. When only choline was added as the sole nitrogen source, activity was found even in the presence of glucose and glycerol.

TABLE V. EFFECT OF CARBON AND NITROGEN FIG. 6. Standard Curve of Choline Estimation. SOURCES ON CHOLINE DEHYDROGENASE The reaction condition was described in the Text. Each compound was added to the basal medium at the concentration of 2% (w/v) except for choline or calculated value from the molar absorbancy NH4Cl was employed as sole nitrogen source at the

index. concentration of 0.5% (w/v). The basal medium consisted of 10 mmoles of KCl, 1 mmole of Na2SO4, The recovery of choline from some biological 0.4 mmole of MgSO4, 40 mmoles of Na2HP04 and materials was investigated. Original amount of 22 mmoles of NaH2P04 in 1 liter deionized water. The

choline in some biological materials such as enzyme activity was assayed in the standard condition. casamino acid, yeast extract, peptone and meat extract was assayed by this method. To each sample, 7.0ƒÊg of choline was added, and then the total content of choline in each sample was determined by this assay method. The re covery of added choline from each sample was examined (Table IV). All materials except yeast extract did not interfere with the assay of choline.

Formation of choline dehydrogenase

Previously we reported the effect of the carbon and nitrogen source on the formation a) : Growth was barely observed . of choline dehydrogenase in P. aeruginosa

A-16 by following the reduction of ferricy DISCUSSION anide.3) However, it has become apparent lately that by the assay method the activity of Since initial studies,1,2,9) the fact that the choline dehydrogenase was not directly meas oxidation of choline to betaine represents the

TABLE IV. EFFECT OF BIOLOGICAL MATERIALS ON THE ESTIMATION OF CHOLINE

The reaction was carried out in the reaction mixture (2.88ml) containing 0.86unit of choline de hydrogenase, 1.3 mmoles of potassium phosphate buffer (pH 7.4), 0.7 ƒÊmole of PMS, 0.43 ƒÊmole of MTT, 0.05 ƒÊmole of choline chloride and each biological materials at 30•Ž for 15min. Absor bancy was measured at 570nm against enzyme blank and the value of choline was determined from the standard curve. Bacterial Choline Dehydrogenase 2083

action of two separate dehydrogenase has been previous result. When the sonication treat made clear. As to the microbial oxidation of ment was prolonged, the reactivity of the

choline, it has been reported that choline was particulate enzyme with ferricyanide tended to directly oxidized to betaine in the intact cells decrease, therefore, the conflict with the previ of Achromobacter cholinophagum.10) A betaine ous result might be due to the fragility of the aldehyde dehydrogenase was found in the cells respiratory chain involved in the particulate. of P. aeruginosa A-l6 grown on choline in the The oxidation of a number of choline anal previous paper.11) The present study showed ogues by rat liver homogenates and mito that choline was oxidized to betaine aldehyde chondrial preparation was examined,12) but the by choline dehydrogenase from P. aeruginosa substrate specificity of the purified enzyme has A-16 by two enzymes, choline dehydrogenase not yet been studied in detail.13) Choline de

and betaine aldehyde dehydrogenase, in the hydrogenase of P. aeruginosa was active to- same way as the mammalian liver. wards only choline among the compounds

About 70% of choline dehydrogenase ac tested, which included alcohols, amino deriva

tivity in extract of P. aeruginosa A-16 was in a tives, and choline analoges. particulate form. The particulate enzyme 2-Dimethylaminoethanol was not oxidized oxidized choline to betaine aldehyde without by the partially purified enzyme, and no requiring any dissociable coenzymes. The oxidizing activity towards 2-dimethylamino- particulate enzyme was found to reduce ethanol was detected even in the crude extract. significantly potassium ferricyanide or PMS in Then, the oxidation of choline in this organism the presence of choline. While, the Triton should occur before the demethylation. treated enzyme showed the formation of be The most widely used method for the quanti taine aldehyde only with the addition of PMS. tative determination of choline is due to the

Potassium ferricyanide no longer functioned as precipitation as reineckate. This method is electron acceptor. The loss of reactivity with relatively non-specific and its sensitivity is very ferricyanide seemed to be a result of the separa low.14) A cholineless mutant of Neurospora tion of the dehydrogenase from the respiratory crassa was used for the microbial method.15•`17) chain. As the activity of the partially purified This method seems to be highly impractical enzyme was not affected by potassium cyanide, because it takes several days for the growth of autooxidizability should be damaged in the the mold. As enzymatic method using [14C]- electron transfer sequence of the particulate acetyl-CoA and choline acetyltransferase was enzyme by the addition of potassium cyanide. recently reported.18) The sensitivity of the Though it is not clear that what kinds of com method is very high but the method requires ponents are involved in the particulate, elec a complicate procedure. The Pseudomonas trons could be transferred to molecular oxygen enzyme studied here was highly specific for by way of cytochrome-like respiratory chain, choline and could be more easily prepared which participation suggested with the dif than the liver enzyme. Then, the application ference spectrum of the particulate. of the enzyme to choline estimation was at In the previous paper," we examined the tempted. It is expected that this enzyme may effect of various carbon and nitrogen sources be useful for the simple and specific analysis on the choline dehydrogenase formation by of choline in blood and foodstuffs and the the assay according to the reduction of ferricy activity of phospholipase D by using the anide. Then choline and acetylcholine were enzyme in the PMS-MTT system. Further effective on the enzyme formation, however, investigation are now in progress. betaine was ineffective on the enzyme forma tion. By assay method using the PMS-DCPIP REFERENCES system, betaine was found to be also effective 1) F. Bernheim and M. L. C. Bernheim, J. Biol. on the enzyme formation in conflict with the Chem., 104, 438 (1933). 2084 T. NAGASAWA, N. Mote, Y. TANI and K. OGATA

2) F. Bernheim and M. L. C. Bernheim, J. Biol. 10) H. S. Shieh, Canad. J. Microbiol., 10, 837 (1964). Chem., 121, 55 (1938). 11) T. Nagasawa, Y. Kawabata, Y. Tani and K. 3) T. Nagasawa, Y. Kawabata, Y. Tani and K. Ogata, Agr. Biol. Chem., 40, 1743 (1976). Ogata, Agr. Biol. Chem., 39, 1513 (1975). 12) T. C. Wells, J. Biol. Chem., 207, 575 (1954). 4) I. M. Armstrong, Biochem. Biophys. Acta, 86, 195 13) T. Kimura and T. P. Singer, "Method in En (1964). zymology," Vol. 5, Academic Press, Inc., New 5) O. H. Lowry, N. J. Rosebrough, A. L. Farr and York, 1962, p. 562. R. J. Randall, J. Biol. Chem., 193, 265 (1951). 14) F. J. R. Beattie, Biochem. J., 30, 1554 (1936). 6) M. Jellinek, D. R. Strength and S. A. Thayer, 15) H. Horowitz and G. W. Beadle, J. Biol. Chem., ibid., 234, 1171 (1959). 150, 325 (1943). 7) R. R. Eady and P. J. Large, Biochem. J., 106, 245 16) H. Horowitz, D. Bonner and M. B. Houlahan, (1968). ibid., 159,145 (1945). 8) W. S. Kristler and E. C. E. Lin, J. Bacteriol., 107, 17) H. Horowitz, ibid., 162, 413 (1946). 1224 (1971). 18) P. A. Shea and M. H. Aprison, Anal. Biochem., 9) P. J. G. Mann and J. H. Quastel, Biochem. J., 31, 56,165 (1973). 869 (1937).