Agric.2663 Biol Chem., 52 (10), 2663~2667, 1988 Rapid Paper as the only product of this enzymatic reduc- tion, we have named this enzyme pseudotro- Purification and Characterization of pine-forming tropinone reductase. Pseudotropine Forming Tropinone Reductase from Hyoscyamus MATERIALS AND METHODS niger Root Cultures Chemicals. Tropine, 2-pyrrolidone, ./V-methyl-2-pyr- rolidone, 4-methylcyclohexanone, and ./V-methylmor- Birgit Drager, Takashi Hashimoto pholine were purchased from Nakarai Chemicals, and Yasuyuki Yamada Kyoto, as were most of the other chemicals used in the buffers and tissue culture media. 7V-methylpiperi- Research Center for Cell and Tissue Culture, dine, vV-methylpiperazine, and 1-methylpiperidine were Faculty of Agriculture, Kyoto University, obtained from WakoPure Chemicals, Kyoto; and tro- Kyoto 606, Japan pinoneand the other tropinone analoguesusedwereob- Received July 21, 1988 tained from Aldrich Chemicals. Pseudotropine and nor- tropinone were gifts from Professor G. G. Gross, Uni- versity of Ulm, West Germany. Apseudotropine-forming tropinone reductase was ex- tracted from root cultures of Hyoscyamusniger that Buffers. Buffer A for extraction: 0.1 m potassium phos- produce the tropane alkaloids hyoscyamine and scopol- phate (pH 7.0), 3 mMdithiothreitol, 0.25 m sucrose. Buffer amine. The enzymestereospeciflcally reduces tropinone to B for column elution and storage: 20mMpotassium phos- pseudotropine, oxidizing NADPH.It has an approximate phate (pH 7.0), l mMdithiothreitol, 0.25m sucrose, 23% molecular weight of 84,000 and a pH optimum between (v/v) glycerol. For the FPLC experiments the glycerol 5.8 and 6.25. The Kmvalue for tropinone is 35.1 /miol/1 content was lowered to 16.7% (v/v). and for NADPH21.1 /imol/1. Substrate specificity was tested for NADPHand several tropinone analogues. Root cultures. Rootcultures of Hyoscyamusniger L., line Hnl 1, were established as described elsewhere.4) They Hyoscyamine and scopolamine are tropane were cultured in 100-ml flasks containing 25ml of B5- alkaloids produced by plants of the Sola- medium5) supplemented with 1 /im indole-3-butyric acid naceae. They are esters formed by tropic acid and 3% sucrose on a gyratory shaker at 90 rpm in the and the secondary alcohol tropine. As wenow dark. Before enzyme extraction the roots were transferred know, tropic acid is derived from phenyl- to 300-ml flasks containing 75 ml of hormone-free medium and cultured for 1 week. alanine by intramolecular rearrangement, and tropine is thought to be formed from ornithine Enzyme extraction. A total of 357g of roots was col- via hygrine and further cyclization to tropi- lected by suction filtration, then frozen in liquid nitrogen none (Fig. 1, for a review see Leete1*). Tropine and homogenized in a Nissei AMblender. Subsequent procedures were done at 4°C. The homogenate was mixed dehydrogenasefrom Datura stramoniumwas with sea sand, 10% (w/v) insoluble polyvinylpyrrolidone, described by Koelen and Gross2) as an enzyme and 500ml of buffer A, then ground thoroughly in a mor- that reduces tropinone stereospeciflcally to tar for 20min. After the mixture was squeezed through tropine. Pseudotropine, the 3jS-hydroxyisomer a 3-layer cheese cloth/Miracloth/cheese cloth filter, the of tropine, was not found as a product. This filtrate was centrifuged at 10,000 xg for 40min. The re- result is consistent with the finding that the sulting supernatant was the crude extract. The tropinone predominant tropane alkaloids in the Sola- reductase in the crude extract was precipitated between 40% to 75% ammoniumsulfate saturation. The precipitate naceae are esters with tropine (i.e., tropane- obtained after 40min of centrifugation at 10,000 x g was 3a-ol); only occasionally have pseudotropine dissolved in 45ml of buffer B. derivatives been described and only as minor Hydrophobic interaction chromatography. The enzyme alkaloids.3) Here we describe the purification solution was adjusted to 30% ammonium sulfate satu- and characteristics of a tropinone-reducing ration and put onto a butyl-Toyopearl column (23x enzymefrom cultured roots of Hyoscyamus 2.3cm) equilibrated with buffer B containing 30% am- niger that accumulate scopolamine and hyo- moniumsulfate saturation. Protein was eluted in steps scyamine. As we have identified pseudotropine with buffer A containing 30%, 20%, 10%, and 0% satu- 2664 B. Drager, T. Hashimoto and Y. Yamada Fig. 1. Proposed Biosynthesis of Tropane Alkaloids. Anarrow mayindicate more than one step. rations of ammoniumsulfate at a flow rate of 1 ml/min. Enzymeassay. Tropinonereductase activity was assayed Each step was continued until the UV-absorption of the by following the change in the light absorbed by NADPH eluted liquid at 280nm was stable. Enzymeactivity was at 340nm and 30°C. The assay mixture (1 ml) contained, eluted with 20% ammoniumsulfate saturation. Active depending on the stage of purification, 2-400 fig of fractions were pooled and passed through a Sephadex G25 protein, 1 fimo\ of tropinone, 200nmol of NADPHand column (41 x 2.8 cm) equilibrated and eluted with buffer B lOOjumol of potassium phosphate buffer, pH 6.25. The containing 0.1 m NaCl. reference contained no tropinone. For the measurementof pHdependency, reaction buffers of the same ionic strength DEAE-chromatography.Enzymesolution adjusted to were used: sodium acetate (pH 4.0-5.5), potassium phos- 0.1m NaCl was put on a DEAE-Toyopearl column phate (pH 5.8 -7.5), Tris-HCl (pH 7.8 -9.0), and glycine- (23.3 x 2.1 cm) equilibrated with buffer B containing 0.1 m HC1(pH 9.3 - 10.0). In tests of substrate analogues tropi- NaCl. After washing the column with 0. 1 m NaCl-buffer B none was replaced by the compounds listed in Table II. In until there was stable UV-absorption, a linear elution the inhibitor test 1 /miol each of tropinone and the pu- gradient of 0.1 to 0.2m NaCl in 800ml of buffer B was tative inhibitor per assay were added. used (flow rate 1 ml/min). Active fractions were pooled and concentrated by Amicon ultra filtration. Reaction product analysis. Assay mixtures of 4 to 10ml were incubated for 2hr. The reaction was stopped by SDS-PAGE gel electrophoresis. Gel electrophoresis precipitating the protein with 20-40ml of acetone. After was done by the method of Laemmli.6) centrifugation, the supernatant was concentrated by evap- oration and lyophilization. The residue was dissolved in Protein measurement.Protein concentrations were mea- methanol and used for TLC, GLC, and GC-MSanalyses. sured by the method of Bradford7} with bovine serum For product analysis the protein solution had first to be albumin (Sigma, fraction V) as the standard. transferred to glycerol-free buffer because glycerol in- terfered with the analysis. GLC was done with a gas Tropinone Reductase from HyoscyamusRoot Cultures 2665 Table I. Purification of Tropinone Reductase from Cultured Roots of Hyoscyamusniger The enzyme activity present after each purification step was tested by the standard assay. Purification step Total protein Specific activity Purification factor Total activity Yield (mg) Okat/kg protein) (-fold) (nkat) (%) Crude extract 890 40 - 75% (NH4)2SO4 420 23.1 9.7 100 Butyl-Toyopearl 93.6 417 39.9 402 DEAE-Toyopearl 1.61 17540 28.2 291 chromatography system (GC7A, Shimadzu Co.) equipped 84,000±4000 (average of 13 measurements). with a capillary column (CBP1 -M25-025, Shimadzu Co.) SDS-PAGEelectrophoreses of the above de- and an FID-detector. Tropinone, tropine, and pseudo- scribed enzyme preparation and of several tropine were analyzed without previous derivatization in a enzyme extracts in our preliminary experi- temperature gradient of 120 ~ 180°C, gaining 5°C/min and having an initial lag time of 2min. GC-MSanalysis was ments showeda strong commonband at the done as described elsewhere.4) TLC on silica gel plates was molecular weight of 39,000, which suggests developed in a solvent system of acetone-ammoniawater that the intact enzymemight be a dimer com- (4: 1). Tropinone, tropine, and pseudotropine were de- tected with Dragendorff reagent, var. Munier,8) which is posed of two identical subunits. especially sensitive for tropine and pseudotropine (de- tection limit 10 nmol/spot). pH and temperature optima, Km-values The highest enzyme activity was found at RESULTS AND DISCUSSION pH 5.8, more acidic buffers greatly inactivated the enzyme (8% of the maximumactivity at Enzymepurification pH 4.5). More alkaline buffers inhibited the Results of the purification procedures are activity less severely (61% of the maximum shown in Table I. In the crude extract, enzyme activity at pH 8.5). This is consistent with activity could not be measured accurately, and findings for other dehydrogenase enzymes that in the ammoniumsulfate fraction the activity have similar pH optima for the reducing was still low. The overall activity yield of 291 % reaction.9) is explained by the removal of an inhibiting The optimum temperature was 30°C. At factor by the butyl column step. After DEAE- 24°C the enzyme showed 77%of its maximum column chromatography the enzymeprepara- activity, and at 56°C was inactivated com- tion could be stored at -20°C for 3 months pletely. and at 4°C for 4 days without loss of activity. The Km value for tropinone was found When kept in only 10% glycerol at 4°C, the at 35.1 /miol/1 and that for NADPH, 21.1 enzyme lost all activity within two days. //mol/1. Koelen and Gross2) reported the pronounced lability of their enzyme, especially upon di- Substrate specificity lution. This prompted us to use a buffer with NADHwas slowly oxidized, but the velocity 23%glycerol which turned out to be necessary was only 5.4% of the velocity found with for enzymestabilization. This enzymeprepara- NADPH.Other dehydrogenase enzymes from tion obtained after DEAE-columnchromatog- secondary product pathways also showed raphy was used to investigate the characteris- marked preferences for NADPH.10~12) tics of the enzyme.