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Acyclic Monoterpene Primary Alcohol :NADP+ Oxidoreductase of Rauwolfia Serpentina Cells: the Key Enzyme in Biosynthesis of Monoterpene Alcohols

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Acyclic Monoterpene Primary Alcohol :NADP+ Oxidoreductase of Rauwolfia Serpentina Cells: the Key Enzyme in Biosynthesis of Monoterpene Alcohols J. Biochem. 109, 341-347 (1991) Acyclic Monoterpene Primary Alcohol :NADP+ Oxidoreductase of Rauwolfia serpentina Cells: The Key Enzyme in Biosynthesis of Monoterpene Alcohols Hiromitsu Ikeda, * Nobuyoshi Esaki, ** Shunji Nakai, * Keij i Hashimoto, Shinichi Uesato,*•' Kenji Soda,** and Tetsuro Fujita*2 *Department of Natural Product Chemistry , Faculty of Pharmaceutical Sciences,Kyoto University,Sakyo-ku, Kyoto, Kyoto 606; **Laboratory of Microbial Biochemistry,Institute for ChemicalResearch, Kyoto University, Uji, Kyoto 611; and *`'Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Kyoto 607 Received for publication, August 1, 1990 Acyclic monoterpene primary alcohol: NADP+ oxidoreductase, a key enzyme in the biosynthesis of monoterpene alcohols in plants, is unstable and has been only poorly characterized. However we have established conditions which stabilize the enzyme from Rauwolfza serpentina cells, and then purified it to homogeneity. It is a monomer with a molecular weight of about 44,000 and contains zinc ions. Various branched-chain allylic primary alcohols such as nerol, geraniol, and 10-hydroxygeraniol were substrates, but ethanol was inert. The enzyme exclusively requires NADP+ or NADPH as the cofactor. Steady-state kinetic studies showed that the nerol dehydrogenation proceeds by an ordered Bi-Bi mechanism. NADP+ binds the enzyme first and then NADPH is the second product released from it. Gas chromatography-mass spectrometric analysis of the reaction products showed that 10-hydroxygeraniol undergoes a reversible dehydrogenation to produce 10-oxogeraniol or 10-hydroxygeranial, which are oxidized further to give 10-oxogeranial, the direct precursor of iridodial. The enzyme has been found to exclusively transfer the pro-R hydrogen of NADPH to neral. The N-terminal sequence of the first 21 amino acids revealed no significant homology with those of various other proteins including the NAD(P)+-dependent alcohol dehydrogenases registered in a protein data bank. Many indole alkaloids produced by Apocynaceae, Logania oxidation to produce 10-oxogeranial through 10-hydroxy ceae, and Rubiaceae are physiologically active and have geranial or 10-oxogeraniol (Scheme 1). been used not only as drugs but also a poisons (1). The tryptamine moiety of indole alkaloids is known to be EXPERIMENTAL PROCEDURES derived from tryptophan (2, 3). The non-tryptamine moiety is a derivative of iridoid (4, 5), which is biosynthe Materials•\Butyl Toyopearl 650M, DEAE Toyopearl sized from acyclic monoterpenes, i.e., geraniol and nerol, 650M, and AF-Red Toyopearl 650ML were purchased from through 10-hydroxygeranioland 10-hydroxynerol,respec Toso Manufacturing, Tokyo; Cellulofine GCL-1000-sf from tively (6- 9). We have established the following biosyn Chisso, Tokyo; NAD+, NADP+, NADH, and NADPH from thetic mechanism for iridoids and the non-tryptamine moiety Kohjin, Tokyo; substrates and inhibitors from Tokyo of indole alkaloids of Rubiaceae and other plants (10-13). Kasei, Tokyo; standard proteins for molecular weight 10-Hydroxygeraniol and 10-hydroxynerol are oxidized to determination from Boehringer Mannheim and Pharmacia the corresponding dialdehydes (i. e. 10-oxogeranial and LKB Biotechnology; iodoacetamide and Polyclar AT from 10-oxoneral), then reductively cyclized to iridodial, which Wako Pure Chemicals, Osaka; and all other chemicals were is finally converted to iridoids (Scheme 1). In fact, a crude of the highest grade commercially available. extract of cultured cells of Rauwolfia serpentina (Apocyna Spectroscopy-UV-visible spectra were recorded in a ceae) (14) oxidizes 10-hydroxygeraniol to produce 10-ox Hitachi 200-20, circular dichroism spectra in a Jasco J-600, ogeranial through 10-oxogeraniol or 10-hydroxygeranial in atomic absorption spectra in a Shimadzu AA-670G, and gas the presence of NADP+ (15). Thus, we have proposed that chromatography-mass spectrometry was performed in a an NADP+-dependent alcohol dehydrogenase acting on Hitachi M80-A equipped with a 003B data system. Isoelec 10-hydroxygeraniol plays an important role in the biosyn tric points were determined with a Pharmacia Phast thesis of the iridoids. System. We here describe the purification of the dehydrogenase Synthesis -Geraniol was acetylated with acetic anhy from R. serpentina cultured cells and its physicochemical dride, then oxidized with selenium dioxide in the presence and enzymological properties. The homogeneous enzyme of tert-butyl hydroperoxide to produce 10-oxogeranyl indeed catalyzes both steps in the 10-hydroxygeraniol acetate, which was hydrolyzed with potassium carbonate in water to give 10-oxogeraniol. 10-Oxogeranyl acetate was 1 Present address: Pharmaceutical Research Laboratories, Japan Tobacco Inc., Yokohama, Kanagawa 227 reduced with sodium borohydride to yield 10-hydroxygera- 2 To whom all correspondence should be addressed . nyl acetate, which was hydrolyzed with potassium carbon- Vol. 109, No. 2, 1991 341 342 H. Ikeda et al. Scheme 1. Biosynthesis of indole alkaloids and iridoids from acyclic monoterpenes in plants. ADH, acyclic monoterpene pri mary alcohol dehydrogenase. ate in water to yield 10-hydroxygeraniol (16). Geraniol was oxidized to geranial with manganum dioxide, then hydrox ylated at C-10 with selenium dioxide to afford 10-hydroxy geranial. Neric acid was prepared from nerol by sodium chlorite oxidation (17) and esterified with diazomethane. Neric acid methyl ester obtained was reduced with [2H4] - lithium aluminium hydride to give [1-2H2]nerol. The C-4 hydrogen of NADP+ was replaced by deuterium from deuterium oxide with potassium cyanide. [4-2H]NADP+ was reduced stereospecifically to (4R) and (4S)-[4-2H]- NADPH with D-glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, respectively; then these prod ucts were purified with DEAE Toyopearl, followed by crystallization as the barium salts (18). ed by centrifugation at 10,000 xg for 30min. Enzyme Assay-The activity of acyclic monoterpene Step 2: Ammonium sulfate precipitation: Ammonium primary alcohol dehydrogenase was determined in a reac sulfate was added to the crude extract at 35% saturation tion mixture (1.0ml) containing 20ƒÊmol of Bis Tris with stirring. The mixture was then centrifuged at Propane HCl buffer (pH9.0), 0.4ƒÊmol of NADP+, 2ƒÊmol 10,000 xg for 30min. The supernatant solution was of 10-hydroxygeraniol, and the enzyme at 25°C. The brought to 70% saturation by further addition of ammo reaction was started by addition of the substrate dissolved nium sulfate. The protein precipitate, collected by centrifu in 10ƒÊl acetone. The NADPH produced was determined at gation at 10,000 xg for 30min, was dissolved in 45ml of 20 340nm. One unit of the enzyme activity was defined as that mM Tris HCl buffer (pH 8.0) containing 20% saturated amount which catalyzes the formation of 1ƒÊmol of NADPH ammonium sulfate. per minute at 25°C. The reverse reaction was followed in a Step 3: Butyl Toyopearl hydrophobic chromatography: reaction mixture (1.0ml) containing 20ƒÊmol of Bis Tris The enzyme solution was applied to a Butyl Toyopearl Propane HCl buffer (pH7.0), 0.1ƒÊmol of NADPH, 1ƒÊmol 650M column (2.5 x 10cm) equilibrated with 20mM Tris of 10-hydroxygeranial, and the enzyme at 25°C. HCl buffer (pH 8.0) containing 1mM dithiothreitol and Protein Determination-Protein was determined by the 20% saturated ammonium sulfate. The column was washed method of Bradford (19). Bovine serum albumin was used with the above buffer. The protein was eluted by decreasing as the standard. Protein was monitored at 280nm during the ammonium sulfate concentration linearly from 20 to purification. 0%. The active fractions eluted with 0% ammonium sulfate Cultivation of Cells-The cell culture of R. serpentina were combined and glycerol was then added to the solution was prepared and provided as a gift by Prof. Dr. M.H. Zenk at a final concentration of 20%. The enzyme solution was of Munich University. Cells were grown in Linsmaier- concentrated by ultrafiltration with an Amicon PM10 Skoog medium containing 1ƒÊM 2,4-dichlorophenoxyace membrane followed by dialysis against the standard buffer. tate and potassium naphthalene acetate for 7 days. They Step 4: DEAE Toyopearl column chromatography: The were collected by filtration through Miracloth, washed with enzyme solution (142ml)was applied to a DEAE Toyopearl distilled water, frozen in liquid nitrogen, then stored at 650M column (2.5 x 7.5cm) equilibrated with 20mM Tris -80°C until use . The frozen cells were kept at -80°C with HCl buffer (pH8.0). The enzyme was eluted by a linear only slight loss of activity for 6 months. increase in NaCl concentration up to 300mM. The active Enzyme Purification-All purification procedures were fractions were combined, dialyzed against 20mM Tris HCl carried out in 20mM Tris HCl buffer (pH8.0) containing 1 buffer (pH 8.0) containing 1mM dithiothreitol, then con mM dithiothreitol and 20% glycerol below 4°C unless centrated to about 20ml with a PM10 membrane. otherwise stated. Step 5: AF-Red Toyopearl dye affinity column chro Step 1: Preparation of crude extract: The frozen cells matography: The enzyme was applied to an AF-Red (383g), suspended in 800ml of 20mM Tris HCl buffer (pH Toyopearl 650ML column (1 x 8cm), which was washed 8.0) containing 1mM EDTA, 1mM dithiothreitol, 1mM with 20mM Tris HCl (pH 8.0) containing 1 mM dithio phenylmethylsulfonyl fluoride, and 20g Polyclar AT, were threitol. The enzyme was eluted with a linear gradient stirred for 3 h in a nitrogen atmosphere and then filtered elution between 0 and 1M KCl. Active fractions were through Miracloth. The crude extract (980ml) was obtain combined and glycerol was then added to the solution at a J. Biochem. Acyclic Monoterpene Primary Alcohol:NADP+ Oxidoreductase 343 final concentration of 20%, then concentrated to 5.2ml with the enzyme from the cell suspension cultures of R. serpen a PM10 membrane. tina. The specific activity of the final preparation was about Step 6: Cellulofine GCL-1000-sf column chromatog 87-fold higher than, that of the crude extract. The purified raphy: The enzyme solution was applied to a Cellulofine enzyme was found to be homogeneous by sodium lauryl GCL-1000-sf column (1.6 x 90cm).
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