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
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. determined in each tube. Assays. Vanillyl alcohol, vanillin and vanillic acid were IR and mass spectra. The IR spectrum and mass spec- determined by high performance liquid chromatography. ATrirotor, Japan Spectroscopic Co., was used with a reverse phase column, JASCOPACK SC-02, Cosmosil 5C18, and equipped with a UV detector, UVIDEC-II, for 15- -1.5 monitoring at 240nm and a digital integrator, System InstrumentsCo., model 5000-E. The mobile phase was 50% methanol at a flow rate of 0.2ml/min.
j/A vK« c O Chemicals. DEAE- and CM-celluloses were purchased }åP^-*§-|.0 -tzlOå -\ / «o from Whatman Chemical Separation Ltd., England. 1:3 \ -/ / \ "° < Sephadex G-200 and Ultrogel AcA 34 were obtained from / r-i ° Germany. Vanillyl alcohol was purified by repeating re- crystallization from hot chloroform solution. Other chemicals used were of reagent grade without further purification.
oi ^+^r , J , ^^^_l-lo RESULTS 0 24 48 72 96 120 Cultural time(hr) roduction of PMS-vanillyl alcohol dehydro- Fig. 1. Production of PMS-Linked Vanillyl Alcohol gen ase
Dehydrogenase and Vanillin Dehydrogenase under P As previously noted,1'2) jR. acidophila M4G2 Aerobic Conditions. produced PMS-vanillyl alcohol and PMS- The organism was grown on 10-liters of medium sup- vanillin dehydrogenases in the medium con- plemented with vanillyl alcohol in a 14-liters Microferm taining vanillyl alcohol. Activities of both en- fermenter at 30°C for 108 hr. The initial concentration of zymes in aerobically grown cells were higher vanillyl alcohol (Val) was 5.0mM. Samples (300-500ml than in cells grown anaerobically. Profiles of of medium) were taken every 12hr. Vanillyl alcohol, vanillin (Van) and vanillic acid (Va) in the medium were the production of these dehydrogenases were determined by HPLC. PMS-vanillyl alcohol dehydro- demonstrated on 10-liters of medium in a jar genase and PMS-vanillin dehydrogenase activities were fermenter under aerobic conditions. As illus- assayed in the crude extracts which were prepared from trated in Fig. 1 , PMS-vanillyl alcohol dehydro- the cells obtained at each interval, and expressed as units in 10 liters of medium. Symbols: à", vanillyl alcohol; O, genase activity appeared after 36-hr incu- vanillin; £, vanillic acid; å , PMS-vanillyl alcohol dehy- bationand reached the maximum after 72 hr. drogenase activity; å¡> PMS-vanillin dehydrogenase ac- PMS-vanillin dehydrogenase activity was also tivity; A, absorbance at 650nm. detected after 60hr incubation and reached 2176 K. Yamanaka and Y. Tsuyuki
the maximum at 72hr. Over 90% of vanillyl (3) Ammonium sulfate fractionation. The alcohol was consumed during 72hr incu- protamine supernatant solution (102ml) was bation. Vanillin and vanillic acid subsequently brought to 30% saturation with ammonium appeared in the medium and were gradually sulfate, and the precipitate was centrifuged dissimilated. at14,000xg for 20min and discarded. Ammonium sulfate was further added to the Enzyme purification supernatant solution to give 80% saturation. All operations were carried out at 5°C, The precipitate was collected by centrifugation unless otherwise specified. and dissolved in 60ml of 50mM Tris-HCl (1) Cell-free extract. Cells (17g, wet weight buffer, pH 7.5. The enzyme solution was dia- from 10-liters of medium) were suspended in lyzed against the same buffer for 12hr. The 50ml of 50mM potassium phosphate buffer, insoluble precipitates formed during dialysis pH 7.5, and disrupted by sonic oscillation at were removed by centrifugation. 20kHzfor lOmin. Intact cells and cell debris (4) Chromatography on DEAE-cellulose. were removed by centrifugation at 14,000 x g The dialyzed enzyme solution (72ml) was ap- for 15 min. Unbroken cells were resuspended plied to a DEAE-cellulose column (2.7 by in 50ml of the same buffer and the sonic 40 cm) equilibrated with 50 mMTris-HCl buf- treatment repeated for a further lOmin in the fer, pH 7.5. The column was washed with same manner. The first and second super- 300ml of the buffer containing 0.1m KC1. natants were combined (cell-free extract). PMS-vanillyl alcohol dehydogenase activity (2) Protamine sulfate. To the cell-free ex- wasrecoveredin this eluate. Thereafter, elu- tract (98 ml), 0. 1 % protamine sulfate solution, tion was carried out with a linear gradient pH 7.0 was added slowly with stirring at the (300ml of 50mM Tris-HCl buffer, pH 7.5, ratio of0.02mg per mg of protein. The pH was containing 0.1 m KC1 in the mixing chamber adjusted to 7.0~7.5. After standing for and300ml of 50mM Tris-HCl buffer, pH 7.5, 20min, the precipitate was removed by cen- containing 0.8 m KC1 in the reservoir; flow rate trifugation and discarded (protamine super- 20ml/hr; each fraction, 4ml). PMS-vanillin natant). dehydrogenase was eluted at about 0.5 m KC1. -6r i3
O YI J I C I 4- I -2O-.1.0
^ I 1 00 §3- ft \ XS-M J> 0/ 16 \ ,' a)
y0 f-x^j100 ^200 3001-|o Fraction number(4ml/tube)
Fig. 2. DEAE-Cellulose Column Chromatography. The experimental details are described in the text. Symbols: O, PMS-vanillyl alcohol dehydrogenase; #, PMS-vanillin
dehydrogenase, -, KC1 concentration; à", absorbance at 280 nm. Vanillyl Alcohol Dehydrogenase of R. acidophila M402 2177
This fraction was active on vanillin with PMS, Effect of pH and temperature on activity but completely inactive on vanillyl alcohol The optimal pH for enzyme activity on withPMS. These two dehydrogenase activities vanillyl alcohol was found to be in a range were produced simultaneously in the cells, but from6.7 to 8.0 in Tris-HCl buffer and max- ould be separated by this procedure (Fig. 2). imum activity was observed at pH 7.5. At pH (5) Gel filtration. The enzyme solution was 6and 9, enzyme activities were 70 and 50% of applied to a Sephadex G-200 column (2.7 by the maximum activity, respectively (Fig. 5). 35 cm) equilibrated with 50 mM Tris-HCl buf- 20-
fer,c pH 7.5, containing 0.1m KC1 and then Q eluted with the same buffer at a flow rate of 12ml/hr. Active fractions were combined and protein was precipitated by adding am- moniumsulfate. Precipitates were collected by -15' I I3 centrifugation and dissolved in the above buf- E u fer. This procedure was repeated once again on Sephadex G-200 column (Step 6). £ !\\ E
a (7) CM-cellulose. The enzyme solution was o \ \\ o SlO- ', -2« placed onto a CM-cellulose column (1.8 by **å o >à" 20cm) equilibrated with 50mM potassium «" ^ > o phosphate buffer, pH 6.5. The column was o "18 ° oj washed with the same buffer and the enzyme activity was recovered in the passed solution. o5 I Jeo -| o Specific activity in each fraction was found to å £ 1 <^ o be almost constant as shown in Fig. 3. A summaryof the enzyme purification is presented in Table I. The enzyme was purified approximately 29-fold from the crude extract q |_ _i ^a ^ |Q g 0 10 k 20 30r of Rhodopseudomonas acidophila M402, and Fraction number the overall yield was 51 %. The purified enzyme showed a single protein band upon disc gel Fig. 3. CM-Cellulose Column Chromatography. electrophoresis. Enzyme activity with vanillyl The experimental details are described in the text. Symbols: O, PMS-vanillyl alcohol dehydrogenase ac- alcohol was found to be located at the same tivity; å , specific activity (v46OOnm/min/^28OnJ; -, ab- position as the protein band (Fig. 4). sorbance at 280nm.
Table I. Purification of Dye-Linked Alcohol Dehydrogenase from Rhodopseudomonas acidophila M402 Dehydrogenase activity was determined with vanillyl alcohol as substrate.
D eh y d r o g en a se T o ta l P u rifica tio n ste p p ro te in S p ec ifi c a ctiv ity Y ield T o ta l u n its (m g ) (% ) ( u n i t s / m g p r o te in ) (1) C e ll-free e x tr ac t 2 ,3 40 7 9 0. 03 4 10 0 (2 ) P ro ta m in e su lfa te 1, 8 70 8 0 0. 04 3 10 1 (3) A m m o n iu m su lfa te fr ac tio n 1,6 6 0 4 4 0. 02 7 5 6 (4 ) D E A E -C e llu lo se 1 3 3 7 6 0. 57 5 9 7 (5) S e p h ad ex G -2 0 0 (1 st) 8 6 6 7 0 .7 80 8 5 (6) S e p h ad ex G -2 0 0 (2 n d ) 6 7 5 0 0. 79 7 6 3 (7 ) C M -C ellu lo se 4 2 4 0 0. 96 3 5 1 2178 K. Yamanaka and Y. Tsuyuki
100r
Fig. 4. Polyacrylamide Gel Electrophoresis of Purified 20- gels as described in the text. AlcoholLower: enzymeDehydrogenase.activity. A sample of the purified protein from the CM-cellulose
s tep (Table I) was electrophoresed in 7.5% polyacrylamide 20 30 40 50 60 Temperature, °C U pper: protein stained with Coomassie brilliant blue. Fig. 6. Effect of Temperature on Activity of Alcohol Dehydrogenase from R. acidophila M402. Activity was measured with vanillyl alcohol at pH 7.0.
0.7-
0.6å "V
> \ o \ 7 8 PH 0.5- \ ig. 5. Influence of pH on Activity of Alcohol Dehydrogenase from R. acidophila M402.
F Activity was measured with vanillyl alcohol as substrate at 30°C. 0.4 - N^
_L The initial velocity at pH 7.5 was measured at 1 1 1 I I 2 4 6 8 10 20 different temperatures from 30 to 60°C. The Molecular Weight(xlO4) maximum activity was observed at 50°C and relatively high activities were observed at Fig.7. Molecular Weight of Alcohol Dehydrogenase fromR. acidophila M402 on Gel Filtration on Ultrogel higher temperatures: 96% at 55°C and 83% at 60°C (Fig. 6). Experimental details were described in the text. 1, myo- globin (MW 17,000); 2, ovalbumin (MW 45,000); 3, bo- Molecular weight vine serum albumin (MW 67,000); 4, y-globulin (MW 160,000) and #, alcohol dehydrogenase. The molecular weight of vanillyl alcohol AcA34. dehydrogenase was determined by the gel fil- tration technique on Ultrogel AcA 34 to be Identification of reaction product from vanillyl 72,000 as shown in Fig. 7. alcohol Vanillin was mostly expected as the product soelectric point of the dehydrogenase reaction from vanillyl
I The isoelectric point of vanillyl alcohol de- alcohol and was confirmed in several ways: (1) hydrogenase as determined by isoelectric spectrum change of the reaction mixture, (2) focusing was pH 6.01 as shown in Fig. 8. detection of new product in the reaction mix-
10 Vanillyl Alcohol Dehydrogenase of R. acidophila M402 2179
à"l"
.--à"à"å å " I'" "'
.à"à"""' I" "* E
Qii« >*' ^ K, -4 -o.i-fe / \ ""^^^""""^ 5 IlZZ^i13Jo ol ___d A_i _J < 10 20 30 40 50 Fraction number(2ml/tube) Fig. 8. Isoelectric Point of Alcohol Dehydrogenase from R. acidophila M402. Experimental details were described in the text. Symbols: O, enzyme activity; à", absorbance at 280nm; pH.
ture by TLC, and (3) analysis of the product 2r : ; : j : ~ ; fter isolation as crystals. Spectral change of the reaction mixture in
thea ultraviolet region was observed by record- ing spectrophotometry. As shown in Fig. 9, absorbance at 340 nm evidently increased dur- ing 60min reaction time. This increase in- dicated the accumulation of the product as vanillin. Vanillin was confirmed in separate experiments. A small portion of the reaction mixture was withdrawn at 1 hr interval, and spotted on a plate of Kieselgel 60 HF254. TLC was developed with a solvent system of benzene-dioxane-acetic acid (90 : 20 : 4, v/v/v). Two spots were always detected for all samples. The lower spot which was detected in the 0-time sample and gradually diminished as the reaction proceeded was identified as vanil- lyl alcohol. The upper new spot having an Rf 250 300 350 400 Wavelength , nm value of 0.42~0.43 corresponded to the Rf value of authentic vanillin. The stoichiometric Fig. 9. Change in Absorbance of Reaction on Vanillyl conversion ofvanillyl alcohol to a product was Alcohol. established as shown in Fig. 10. The product Reaction conditions were described in the text. was analyzed by HPLC and found to be vanillin. Vanillin was accumulated stoichio- vanillyl alcohol (190mg), 1.25mmol PMS, metrically in the reaction mixture. 1.5mmol NH4CI were dissolved in 100ml of Further experiments were carried out to 50him Tris-HCl buffer, pH 7.5, and the pu- identify the reaction sequence of this enzyme. rified enzyme was added (50 units, prepared to As the reaction mixture (100ml), 1.25mmol step 7 in Table I from another batch of 10-liter 2180 K. Yamanaka and Y. Tsuyuki
10-
8å \
"E "E 7 ^^ >>^ / o-o o
0 2 4 6 8 10 50 Reaction time, hr m/e ig. 10. Stoichiometry of Dehydrogenase Reaction on Fig.ll. Mass Spectrum of the Reaction Product. Vanillyl Alcohol.
F O, vanillyl alcohol remaining and #, vanillin formed in C,63.23; H, 5.32; N, 0.00% Calcd for C8H8O3: C, 63.15; H, 5.30; N, 0.00%. (3) IR spectrum: IR spectra of the sample (A) and authentic culture). The reaction proceeded for 4hr at vanillin (B) were identical in all aspects. (4) 30°C. Yanillyl alcohol was completely exhaust- Mass spectrum: The mass spectrum (direct ed at the end of the incubation. The reaction inlet) was shown in Fig. 1 1. The molecular ion was stopped by adding 1.0ml of 10% tri- at m/z was 152 (base ion) and there were the chloroaceticacid, then the mixture was centri- following diagnostic fragment ions; 151, 138, fuged and the supernatant was concentrated in 124, 123, 109 and 81. m/z 152 (H+) suggested vacuo to dryness. Solids were extracted with the molecular formula to be C8H8O3, which 20ml of benzene three times. Solids were fur- has been confirmed by the elemental analysis. ther subjected to four times successive extrac- This formula was also supported by the occur- tion with 50ml of chloroform-water (1 : 1, rence of isotopes, m/z 154 (M+2) and 153 v/v). All of the soluble fractions were com- (M+l), 1.1% (theoretical 0.95%) and 9.25 bined and evaporated to dryness in vacuo (theor. 8.89%), respectively. (5) NMR spec- (yield, 338 mg). Solids were dissolved in 3ml of trum: Chemical shifts in the NMR spectrum of chloroform and submitted to preparative TLC this crystals were observed and assigned at S on Kieselgel60 HF254 (8 plates, 20 by 20cm). 3.91 (3H, s, -OCH3), d 6.96 (1H, d aromatic TLC was developed with same solvent system H), 3 7.40 (2H, d aromatic H) and d 9.78 (1H, asdescribed above. After the band corre- s, -CHO. This NMR spectrum was completely sponding to the new product was detected by identical to that of authentic vanillin. In con- UV, this band was collected and extracted 5 clusion, the reaction product from vanillyl times each with 20ml of chloroform. These alcohol with the alcohol dehydrogenase was extracts were combined and evaporated to identified dryness and crystallized from hot chloroform. The resulting crystals (180 mg) were recrystal- Substrate specificity lized from hot benzene-hexane (3 : 1, v/v) to The substrate specificity of the enzyme was give pure needle-shaped crystals (79.6 mg). determined with thirty-seven compounds (aro- The crystals were subjected to analysis by matic and aliphatic alcohols and aldehydes). the following methods. (1) Melting point: The Enzyme activity was expressed as relative ac- melting point of crystals was 80~81.2°C (3 tivity to the activity on vanillyl alcohol being determinations, uncorrected). It coincided taken as 100. These results are summarized in with that of authentic vanillin (80~81.2°C, Table II. This enzyme was active on vanillyl uncorrected). No drop in melting point was alcohol, but also active on some aliphatic al- observed in the mixed test with authentic cohols, «-propanol and w-butanol at almost vanillin. (2) Elemental analysis: Found: the same level. Activities were also found on
100150
as vanilin. Vanillyl Alcohol Dehydrogenase of R. acidophila M402 2181
able II. Substrate Specificity of Table III. Km Values
T Alcohol Dehydrogenase K m Su bstrate R elative (i"M ) S ub strate activity Rf v alue" (% ) V anillyl alco hol 113 B enzyl alco hol 7 V anillyl alcoho l 100 0.242 2-P hen yl ethan ol 12 B enzyl alcoho l 49 0.240 2-Phenoxy ethan ol 20 C in nam yl alco hol 40 0.241 rc-P ro pan ol 180 Phenoxy ethan ol 4 0 0.236 ｫｰB utano l 14 P h en yl eth ano l 38 0.244 w o-B utano l 130 M eth ano l 0 r-C inn am ald eh yde 258 E th ano l 2 3 0.241 /7-D im ethylam in obenzaldehyd e 300 2-Methoxy ethan ol 0 F orm aldehyd e 9 8 ｫｰP rop ano l 115 0.244 P ro pio nald eh yde 28 5 /so-P ro pano l 0 B utyraldehyd e 10 5 H-B u tan ol 9 1 0.248 w o-B utanol 58 0.245 seo B u tan ol 0 formaldehyde, propionaldehyde and butyral- tert-B u tan ol 0 2-M ethyl b utano l 50 0.240 dehyde, but was inert on acetaldehyde. As to w-P en tan ol 53 0.238 the aromatic aldehydes, the enzyme showed /.w -P entanol 53 0.241 affinity for Jra/w-cinnamaldehyde, but not for sec-P en tan ol 0 vanillin or syringaldehyde. All of these activ- te r/-P en tan ol 0 ｫｰH exan ol 34 0.241 ities were demonstrated as single bands hav- sec-H exan ol 2 7 0.242 ing the same relative mobility of 0.24 on gel ｫｰH ep tan ol 17 0.241 electrophoresis on polyacrylamide. Although ｫｰO ctan ol 15 0.244 the enzymeshowed broad substrate specificity, ｫｰN on an ol 0 H-D ecanol 0 the enzyme was specifically inactive on meth- anol,vanillin, isovanillin and o-vanillin. The C ycloh exan ol 0 1,2-Butane d iol 14 0.239 following compounds did not serve as sub- F orm aldehyd e 61 0.237 strates for the enzyme: Phenol, catechol, o-, m- A cetald eh yde 8 0.241 and p-cresols, 2,4-, 2,5-, 2,6-, 3,4- and 3,5- P ro pio nald eh yde 60 0.240 B utyraldehyd e 64 0.248 dimethylphenols, methylamine, dimethyl- /-C inn am ald eh yd e 4 1 0.247 amine, trimethylamine, diphenylamine and Syrin galdehyde 0 a-naphthylamine. A comparison of the Km V an illin 0 w o-V an illin 0 values on substrates is shown in Table III. 0-V anillin 0 Whereas the enzyme showed higher affinity on 1 , 2-C yclohexane dio l 0 benzyl alcohol than on vanillyl alcohol, aro- matic alcohol dehydrogenase or benzyl alcohol Enzyme activity was detected on the gels after dehydrogenase would be a more suitable name staining with respective substrates. Each band was recorded with a spectrophotometer and expressed as for this enzyme. However, the enzyme also the Rf value. See the text for details of activity showed affinity toward some of aliphatic al- staining. cohols and their aldehydes. Judging from these results, it is better that this enzyme is classified benzyl alcohol, cinnamyl alcohol and other as a new member of the dye-linked alcohol aliphatic alcohols from C5 to C8. Some de- dehydrogenases. rivatives of aliphatic alcohols also served as substrates: Phenoxyethanol, phenyl ethanol, DISCUSSION iso-butanol, wo-pentanol, 2-methyl butanol and sec-hexanol. The enzyme showed equal The enzyme isolated from Rhodopseudo- activity on aliphatic aldehydes, such as monas acidophila M402 is new type of alco- 2182 K. Yamanaka and Y. Tsuyuki hoi dehydrogenase and differs from hitherto cidate the similarity and dissimilarity of the reported primary alcohol dehydrogenase, sec- two enzymes in respect to the reaction mech- ondary alcohol dehydrogenase, diol dehydro- anism and structure of the active center of genase and aromatic alcohol dehydrogenase. these enzyme proteins with reference to the This dehydrogenase activity was demonstrat- substrate specificity. ed in cells grown on vanillyl alcohol, but Duine et al. showed the participation of a the final preparation purified from vanillyl newelecton acceptor, pyrrolo-quinoline qui- alcohol-grown cells was active on various none (abbreviated as PQQ)20~22) or meth- aromatic and aliphatic alcohols such as ali- oxatin,23'24) in methanol (primary alcohol) phatic alcohols from C2 to C8, vanillyl alco- dehydrogenase. PQQ can be demonstrated hol and cinnamyl alcohol. Among these alco- in alcohol dehydrogenase and glucose de- hols, the simplest aromatic alcohol substrate hydrogenase from a non-methylotroph, is benzyl alcohol. Aromatic alcohol dehydro- Acinetobacter calcoaceticus,25'26) methylamine genase which acts on benzyl alcohol has been dehydrogenase from Pseudomonas AM127) reported in Pseudomonas sp.,9) Pseud, alcali- and glucose dehydrogenase from Pseud. genes,16) and Bacterium,17) but little infor- fluorescens.28)Therefore, participation of PQQ mation was available on the activity on vanillyl in the aromatic alcohol dehydrogenase re- alcohol. m-Hydroxybenzyl alcohol dehydro- action is most expected. Since R. acidophila genase has been detected in Pseud. putida10) M402 was isolated as a methanol-utilizing and Penicillium urticae.18) These enzymes were phototrophic bacterium, methanol dehydro- also active on benzyl alcohol, but no activity genase was produced in the methanol-grown was demonstrated on vanillyl alcohol. All of cells under anaerobic conditions.1* This pro- these benzyl alcohol dehydrogenase activities duction has already been observed for R. were dependent on nicotinamide adenine di- acidophila 10050 by Bamforth and Quayle.29) nucleotide. Another type of aromatic alcohol It is supposed that PQQ or related com- dehydrogenase was found in Neurospora cras- pound(s) could possibly be produced by R. sa,19) but this activity together with the en- acidophila M402 in cells grown aerobically zyme from Pen. urticae18) were specific for r anaerobically on vanillyl alcohol. nicotinamide adenine dinucleotide phosphate. Methanol dehydrogenase from R. acidophila Therefore, the enzyme reported in this paper is 10050 was produced in cells grown anaerobi- substantially different from all known benzyl cally in the light on methanol,29) but the same
alcohol dehydrogenases hitherto reported. enzymeo was also detected in the cells grown One of the remarkable feature of this enzyme aerobically on ethanol.30) Bamforth and is its broad substrate specificity. Primary al- Quayle named on this enzyme dye-linked al- cohol dehydrogenase found in methylotrophic cohol dehydrogenase. The alcohol dehydro- bacteria is characterized by having broad sub- genase from R. acidophila M402 was produced strate specificity on aliphatic alcohols, but no from both cells grown aerobically and activity was observed on any of the aromatic anaerobically. While vanillyl alcohol is one of alcohols.5~8) The difference in the substrate the active substrates for the enzyme produc- specificity was evident on methanol. Methanol tion, the vanillyl alcohol-induced dehydro- is the most active substrate for the primary genase is active on a wide variety of alcohols alcohol dehydrogenase, while the alcohol de- including vanillyl alcohol and aliphatic al- hydrogenase in this paper was completely in- cohols. Therefore, we consider the most suit- ert on methanol. However, activities of both able name for this enzyme is dye-linked al- dehydrogenases are dependent on phenazine cohol dehydrogenase rather than vanillyl al- methosulfate, and nicotinamide adenine dinu- cohol dehydrogenase or aromatic alcohol de- cleotide does not serve as an electron accep- hydrogenase. Some measures should be taken tor. It is, therefore, of great interest to elu- to distinguish the two enzymes, e.g. to give a Vanillyl Alcohol Dehydrogenase of JR. acidophila M402 2183
proper name.With our present knowledge, we O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. propose that Quayle's enzyme should be called Randall,/. Biol. Chem., 193, 265 (1951). B.J.Davis,Ann. N. Y. Acad. ScL, 121, 404 (1964). dye-linked alcohol (aliphatic) dehydrogenase K. Yamanaka and K. Matsumoto, Agric. Biol. and ours dye-linked alcohol (non-specific) Chem., 41, 467 (1977). dehydrogenase. P.Andrews,Biochem. J., 91, 222 (1964). C.L. Poh and R. C. Bayly, /. Bacteriol, 143, 59 Acknowledgments. This work was supported by a (1980). Grant-in-Aid for Special Project Research (Production of A. Livingstone and C. A. Fewson, Biochem. J., 130, Useful Materials) from the Ministry of Education, Science 937 (1972). and Culture of Japan, for which the authors are very P. I. Forrester and G. M. Gaucher, Biochemistry, ll, grateful. 1108 (1972). G. G. Gross and M. H. Zenk, Eur. J. Biochem., 8, REFERENCES 420 (1969). J. A. Duine, J. Frank and J. Westerling, Biochim. 1) K. Yamanaka, M. Moriyama, R. Minoshima and Y. Biophys. Ada, 524, 277 (1978). Tsuyuki, Agric. Biol. Chem., 47, 1257 (1983). J. A. Duine and J. Frank, Jr., Biochem. J., 187, 221 2) K. Yamanaka and Y. Tsuyuki, Agric. Biol. Chem., (1980). 47, 1361 (1983). J. A. Duine, J. Frank, Jr. and P. E. J. Verwiel, Eur. J. 3) J. R. Quayle, Adv. in Microbial Physiol., 7, 119 Biochem., 108, 187 (1980). . (1972). S. Forrest, S. A. Salisbury and C. C. Kilty, 4) J. Colby, H. Dalton and R. Whittenbury, Ann. Rev. Biochem. Biophys. Res. Commun., 97, 248 (1980). Microbiol, 33, 481 (1979). T_ Mincey, J. A. Bell, A. S. Mildvan and R. H. 5) C. Anthony and L. J. Zatman, Biochem. J., 96, 808 Abeles, Biochemistry, 20, 7502 (1981).
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