Characterization of Choline Dehydrogenase from Pseudomonas Aeruginosa A-16
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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.