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Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas Acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity

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Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas Acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity Agric. Biol Chem., 47 (10), 2173~2183, 1983 2173 A NewDye-Linked Alcohol Dehydrogenase (Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity Kei Yamanaka and Yasutaka Tsuyuki Institute of Applied Biochemistry, and Graduate School of Master's Program in Environmental Sciences, The University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki 305, Japan Received October 19, 1982 A new dye-linked alcohol dehydrogenase (vanillyl alcohol dehydrogenase) was purified to homogeneity from cells of Rhodopseudomonas acidophila strain M402 grown aerobically on vanillyl alcohol. The reaction product from vanillyl alcohol was identified as vanillin as judged by its melting point, elemental analysis and IR, mass and NMR spectra. The molecular weight of the enzyme was estimated to be approximately 72,000 as determined by gel filtration and the isoelectric point was pH 6.01. The most characteristic feature of this enzyme is its wide substrate specificity range. The enzyme catalyzes the dehydrogenation of various aromatic and aliphatic alcohols and aldehydes with phenazine methosulfate as electron acceptor. The active substrates of this enzyme are as follows: Vanillyl alcohol, benzyl alcohol, cinnamyl alcohol, 2-phenylethanol, 2-phenoxyethanol, aliphatic alcohols of C2 to G8, rr<m?-cinnamaldehyde, formaldehyde, propionaldehyde and butyraldehyde. The highest activities were obtained with vanillyl alcohol, rc-propanol and ^-butanol at the same level. The apparent Kmvalues were as follows: 112/zm for vanillyl alcohol, 7/jm for benzyl alcohol, 180/im for «-propanol, 14^m for #-butanol, 20/zm for 2-phenoxyethanol, 12/iM for 2-phenylethanol and 105 /zm for butyraldehyde. These activities were confirmed to be catalyzed by a single enzyme by activity staining on polyacrylamide gels. But the enzyme is completely inert on vanillin and methanol. Therefore, dye-linked alcohol dehydrogenase is the most suitable name for this enzyme from R. acidophila M402 rather than vanillyl alcohol dehydrogenase or aromatic alcohol dehydrogenase, but this enzyme is substantially different from the dye-linked alcohol dehydrogenase from R. acidophila 10050 which is active on methanol. In previous studies, we reported the ability demonstrated in the presence of phenazine of utilization of vanillyl alcohol, vanillin and methosulfate. As a phenazine methosulfate- vanillic acid of a newly isolated purple non- linked enzyme, methanol dehydrogenase sulfur phototrophic bacterium, Rhodopseudo- (primary alcohol dehydrogenase) has been monas acidophila strain M402.1} The dehydro- reported from many methylotrophic bacte- genase activities on vanillyl alcohol and ria.3>4) This enzyme is well recognized as one vanillin were demonstrated. These activities that catalyzes on its own the oxidation of a were detected in cells grown on vanillyl al- wide range of primary alcohols, from Cx to cohol or vanillin under aerobic-dark or anaer- C10, and some of their derivatives, but does obic-light conditions. We postulated the par- not act on aromatic alcohols such as benzyl ticipation of these dehydrogenase-like en- alcohol.5~8) Katagiri et al. reported the ben- zymes in the degradation of vanillyl alcohol by zylalcohol dehydrogenase from Pseudomonas R. acidophila M402.2) The most active dehy- sp.9) This enzyme required NAD+ as cofac- drogenase activity on vanillyl alcohol was tor. Keat and Hopper found m-hydroxyben- 2174 K. Yamanaka and Y. Tsuyuki zyl alcohol dehydrogenases in />-crosol or 3,5- added as the single carbon source. The pH was controlled xylenol-grown cells of Pseudomonas putida.10) between6.8and 7.2. The culture was carried out at an The enzymes were active both m- and p- aeration rate of 6 liters/min and stirred at 400 rpm at 30°C. The culture was harvested by centrifugation at the end hydroxybenzyl alcohols and other aromatic of the logarithmic growth phase (consumption of vanillyl alcohols including benzyl alcohol. But vanillyl alcohol reached 90%). alcohol was not tested as a substrate for these enzymes. These enzymes were also spe- Enzyme assay. Phenazine methosulfate*-linked dehy- cific for NAD+. Therefore, aromatic alco- drogenase activities were assayed by the decrease in ab- hol dehydrogenases hitherto reported are sorbance at 660nm according to the method employed for the assay of methanol dehydrogenase by Anthony specific for NAD+, and an aromatic alcohol and Zatman.xl) The standard assay mixture (2.0ml) was dehydrogenase which is active on vanillyl al- composed of lO/imol vanillyl alcohol (or an other sub- cohol has not yet been reported. strate), 50^mol Tris-HCl buffer, pH 7.0, 30/imol In the present paper, we describe the purifi- NH4C1, 2.4/miol KCN, 2Jumol PMS, 1.4/miol DCPIP cation of a new dehydrogenase which is active and the enzyme solution. The reaction mixture was maintained at 30°C with a thermostated spectrophotom- on vanillyl alcohol from vanillyl alcohol- eter. One unit of enzyme activity was defined as the grown cells of R. acidophila M402. The pu- amount of enzyme that catalyzed the dehydrogenation rified single enzyme shows broad substrate of one /rniol of substrate per min. Protein was deter- specificity including aromatic and aliphatic mined by the method of Lowry et al}2) using bovine alcohols.Thus, we propose to name this en- serum albumin as the standard. For most column frac- tions, the protein elution patterns were estimated by the zyme dye-linked alcohol dehydrogenase rather absorbanceat 280 nm. than vanillyl alcohol dehydrogenase or aro- matic alcohol dehydrogenase. We also de- Polyaerylamide gel electrophoresis. Disc gel electro- scribe the biochemical properties of this phoresis in a 7.5% polyacrylamide gel was performed by enzyme. the procedure of Davis.13) The enzyme was run at a constant current of 3 mA per tube for 50min. The protein was stained with 0.25% Coomassie brilliant blue in acetic MATERIALS AND METHODS acid-methanol (46 : 454) for 60 min. The gel was destained electrophoretically and stored in 7.5% acetic acid. Microorganism. Rhodopseudomonas acidophila, strain Dehydrogenase activity in the gels was stained by a M402, was used in this experiment.1* It was isolated from modification of the method used for methanol dehydro- surface water of an acidic hot spring at Sugayu, Aomori genase.14) The extruded gels were immersed in small test Prefecture. Strain M402 is capable of growth with vanillyl tubes of a solution (2.3ml) containing 1.2ml of 50mM alcohol,vanillin, vanillic acid and protocatechuic acid as a Tris-HCl buffer, pH 7.5, 0.1 ml of 50mM vanillyl alcohol single carbon source and electron donor under anaerobic- orothersubstrates, 0.02ml of 10mM PMS, 0.08ml of light conditions and with vanillyl alcohol under aerobic- 50mM nitroblue tetrazolium chloride, 0.1 ml of 300mM dark conditions. NH4C1 and 0.8 ml ofdeionized water at 30°C for 20min in thedark. After the incubation, gels were washed with a Cultivation of the organism. The cells were grown aerobi- mixture of acetic acid-methanol-water (70 : 50 : 875, v/v/v) cally at 30°C on a medium containing 0.05% KH2PO4, and stored in 7.5% acetic acid in the dark. Seventeen 0.1% K2HPO4, 0.08% (NH4)2SO4, 0.012% MgSO4- 7H2O, alcohols (five aromatic and twelve aliphatic alcohols) 0.05% yeast extract and 0.1% vanillyl alcohol. Vanillyl and six aldehydes (two aromatic and four aliphatic alde- alcohol was sterilized separately by filtration through a hydes) were used as substrates. Gels were scanned at membrane filter (pore size, 0.45/^m; Millipore Corp., 580nm and recorded with a Gil ford spectrophotometer, Bedford, Mass.). The pH of the medium was adjusted to model 240, equipped with a linear transport, and relative 7.0. R. acidophila M402 was inoculated into 500ml basal mobilities of activity bands were calculated. medium with 1 % acetate as the carbon source and cultured anaerobically in the light at 30°C for 5 days. This culture Molecular weight determination. The molecular weight was then inoculated into 10 liters basal medium in a 14- of vanillyl alcohol dehydrogenase was determined by the liter Microferm fermentor (New Brunswick Scientific Co., method of Andrews15) using a column (1.5 by 89cm) of Inc.,New Brunswick, New Jersey). Vanillyl alcohol was Ultrogel AcA34 which had been equilibrated with 50mM Abbreviations: PMS, phenazine methosulfate; DCPIP, dichlorophenol indophenol; TLC, thin layer chromatog- raphy; HPLC, high performance liquid chromatography. Vanillyl Alcohol Dehydrogenase of R. acidophila M402 2175 Tris-HClbuffer, pH 7.0, containing 0.1 m KC1. Elution tram were measured with a Hitachi infrared spectropho- was carried out at a flow rate of lOml/h. The marker tometer, model 260-30, and a Hitachi Mass spectro- proteins used were as follows: y-globulin, 160,000; bovine photometer, model RMU-6M, respectively. Elemental serumalbumin, 67,000; ovalbumin, 45,000; and myo- analysis and NMR were done at the Analytical Center globin, 17,000. of this University. Isoelectric focusing. Isoelectric focusing of the enzyme Spectrophotometric determination. The dehydrogenation was conducted in a 110-ml column LKB model 8101 reaction on vanillyl alcohol in the presence of PMS was (Bromma, Sweden). Carrier Ampholite (1%, w/v) was recorded with a Hitachi model 220A recording double established with a pH gradient of from 3 to 10. The beam spectrophotometer. The reaction mixture (2.0ml) column was prepared according to the LKB manual. The contained 50/anol Tris-HCl buffer, pH 7.0, 1 /imol PMS, focusing was performed at a constant voltage of 350V for 1 pmo\ vanillyl alcohol, 30^mol NH4C1 and the purified 48hrat 2°C. Fractions of 2ml were collected from the enzyme preparation in a 3-ml quartz cuvette. The reaction bottom of the column at a flow rate of 40ml/hr. The was followed by repeating the scanning of the spectrum enzyme activity, pH and absorbance at 280nm were from 380nm at 3-min intervals at 30°C.
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