hyoscyamine6f3-hydroxylase, 2-oxoglutarate · pdf filehyoscyamine6f3-hydroxylase,...

Plant Physiol. (I1986) 81, 619-6250032-0889/86/81/061 9/07/$0 1.00/0

Hyoscyamine 6f3-Hydroxylase, a 2-Oxoglutarate-DependentDioxygenase, in Alkaloid-Producing Root Cultures

Received for publication December 24, 1985

TAKASHI HASHIMOTO AND YASUYUKI YAMADA*Research Centerfor Cell and Tissue Culture, Faculty ofAgriculture, Kyoto University, Kyoto 606, Japan

ABSTRACT

Root cultures of various solanaceous plants grow well in vitro andproduce large amounts of tropane alkaloids. Enzyme activity that convertshyoscyamine to 6,6-hydroxyhyoscyamine is present in cell-free extractsfrom cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine6,6-hydroxylase was purified 3.3-fold and characterized. The hydroxyl-ation reaction has absolute requirements for hyoscyamine, 2-oxoglutar-ate, Fe2" ions and molecular oxygen, and ascorbate stimulates thisreaction. Only the L-isomer of hyoscyamine serves as a substrate; D-

hyoscyamine is nearly inactive. Comparisons were made with a numberof root, shoot, and callus cultures of the Atropa, Datura, Duboisia,Hyoseyamus, and Nicotiana species for the presence of the hydroxylaseactivity. Decarboxylation of 2-oxoglutarate during the conversion reactionwas studied using [1-'4Cj-2-oxoglutarate. A 1:1 stoichiometry was shownbetween the hyoscyamine-dependent formation of CO2 from 2-oxoglutar-ate and the hydroxylation of hyoscyamine. Therefore, the enzyme can beclassified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-).Both the supply of hyoscyamine and the hydroxylase activity determinethe amounts of 6,B-hydroxyhyoscyamine and scopolamine produced inalkaloid-producing cultures.

Tropane alkaloid molecules are characterized by the presenceofa bicyclic amine tropane ring system. Although these alkaloidsare distributed in several families that are not related taxonomi-cally, tropic acid esters of hydroxytropane derivatives (e.g. hyos-cyamine and scopolamine) are restricted to Solanaceae speciesAtropa, Datura, Duboisia, Hyoscyamus, and Scopolia (3, 18).The biosynthesis of tropane alkaloids in plants has been investi-gated extensively with labeled precursors (for a recent review, seeLeete [14]). Ornithine is incorporated into the pyrrolidine ringof tropine, whereas tropic acid is formed by the intramolecularrearrangement of thQ side chain of phenylalanine. Tropine andtropic acid then condense to give hyoscyamine. Scopolamine isformed from hyoscyamine via 6(-hydroxyhyoscyamine (19) (Fig.1). 63-Hydroxyhyoscyamine has been isolated from several so-lanaceous plants (4, 11, 18).None of the exact biosynthetic reactions or enzymes that

function in the oxidative conversion of hyoscyamine to scopol-amine are known. Fodor et al. (5) reported that when a solutionof 6,7-dehydrohyoscyamine was fed to almost alkaloid-freescions of Datura ferox L. grafted on Cyphomandra betacea cvSendtn., scopolamine could be isolated from the Datura scionsafter a week (Fig. 1). On the basis of that report, Waller andNowacki (22) speculated that hyoscyamine is first dehydrogen-ated to 6,7-dehydrohyoscyamine which then is converted via 6j3-hydroxyhyoscyamine to scopolamine. By contrast, Fodor et al.(5) and Leete (14) suggested a biosynthetic pathway in which

6,7-dehydrohyoscyamine is an intermediate between 6f3-hydrox-yhyoscyamine and scopolamine; but, this hypothetical unsatu-rated intermediate has yet to be isolated from plants.

Previously (1 1), we reported the successful establishment ofroot cultures of several alkaloid-containing solanaceous plants.These cultured roots grow vigorously when the culture mediumis supplemented with IBA,' and alkaloid biosynthesis is stimu-lated after transfer of these roots to auxin-free medium. Rootcultures of Hyoscyamus niger L. have notably high ratios ofscopolamine to hyoscyamine. The remarkable ability of theroot cultures of this species to synthesize scopolamine was dem-onstrated in an experiment in which a large proportion of anexogeneous supply of hyoscyamine was converted to scopola-mine (10). Thus, root cultures ofH. niger appear to be of use forstudies of the biosynthetic pathway from hyoscyamine to sco-polamine.We here report the isolation and partial characterization of an

enzyme which converts hyoscyamine to 6,B-hydroxyhyoscyaminein root cultures of H. niger. The general involvement of thisenzyme in scopolamine biosynthesis is demonstraled in variousalkaloid-producing and nonproducing cultures.

MATERIALS AND METHODS

Chemicals. L-Hyoscyamine hydrobromide purchased fromNakarai Chemical Co., Kyoto was recrystallized from etha-nol-ether before use. Polyclar AT obtained from Kasei-hinShyouji Co., Osaka was washed with HCI. The 2-oxoglutaricacid, L-ascorbic acid sodium salt and Univer-Gell II were pur-chased from Nakarai Chemical Co., and DTT, FeSO4 7H20 and(-)-(R)-camphor-10-sulfonic acid (monohydrate) from WakoPure Chemicals, Kyoto. The Sephadex G-25 and PD-10 columnswere from Pharmacia Fine Chemicals; the Silica gel 60 PF254,Extrelut- 1 columns, and Extrelut packings from Merck; and the[1-'4C02]-2-oxoglutarate (54 mCi/mmol) and the NCS reagentfrom Amersham. Miracloth was purchased from Calbiochem,and BSA (Fraction V), atropine, and catalase C- 100 from Sigma.Authentic (-)-6p3-hydroxyhyoscyamine, a gift from Dr. Her Liyiof the Chinese Academy of Medical Sciences, was recrystallizedfrom ethanol-ether.D-Hyoscyamine hydrobromide was prepared by modifying the

method of Werner and Miltenberger (23). In short, mixingatropine (D,L-hyoscyamine) and (-)-(R)-camphor-IO-sulfonicacid in acetone gave crystals of (+)-hyoscyamine-camphor-10-sulfonate; m.p. 160°C, [a]25 = +4.0 (c = 18.04, H20). Thediastereomer was converted to D-hyoscyamine, then to D-hyos-cyamine hydrobromide; m.p. 152°C, [a]25 = +24.8 (c = 5.48,H20). Recrystallized commercial L-hyoscyamine hydrobromidegave m.p. 153°C and [a]2 = -24.8 (c = 5.44, H20).

Root Cultures. Root cultures of Hyoscyamus niger L., line

' Abbreviations: IBA, indolebutyric acid; NAA, naphthaleneaceticacid; TMS-derivatives, trimethylsilyl derivatives.

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HASHIMOTO AND YAMADA

N-Me

H

°C-CH-110 CH20O

Z-Hyoscyami

N Me

H 0 CH20H3iine 6 ~-Hydroxyhyosc)

Tropic Acid Tropine

i 4I II I

Phenylalanine Ornithine

N`Me

mm -H

D/ °',C-CH -.I 0 CH20H

yamine Scopolamine

N-Mev

H0 CH-CH

O CH20H

FIG. 1. Metabolic pathway of hyoscyamine to sco-polamine. Hyoscyamine is converted to scopolaminevia 6,-hydroxyhyoscyamine. 6,7-Dehydroxyhyoscy-amine is another hypothetical precursor of scopola-mine (5, 14, 22). See text for details.

6, 7-Dehydrohyoscyamine

Hn 11, established as described elsewhere (11), were subculturedon a Gyrotory shaker (model G 10-21, New Brunswick Scientific,NJ) at 90 rpm and 25°C in the dark in 100-ml flasks containing25 ml of liquid LS (15) or B5 medium (8) supplemented with3% (w/v) sucrose and 1 ,uM IBA. Prior to the experiments, thecultured roots were transferred to 300-ml flasks containing 75ml of medium without auxin, then cultured under the aboveconditions for 1 to 2 weeks.

Extraction and Partial Purification of the Enzyme. The cul-tured roots were frozen with liquid N2 then homogenized in aWaring Blendor. The frozen homogenate was kept at -20°Cuntil use.

Subsequent procedures were done at 4°C. The cell homogenatewas mixed with Polyclar AT (1:1 v/v), then 2 ml/g of cellhomogenate was suspended in 100 mm of K-phosphate (pH 7.5),that contained 0.25 M sucrose and 3 mM DTT. This suspensionwas kept at 4°C for 30 min with occasional stirring, after whichit was passed through a composite cheesecloth-Miracloth-cheese-cloth filter then centrifuged at 13,000g for 20 min. In someexperiments, this supernatant was passed through a SephadexPD-10 column and used as the "crude enzyme preparation."The clear supernatant was fractionated with solid (NH4)2SO4.

Protein that precipitated between 60 to 80% (w/v) (NH4)2SO4saturation was collected by centrifugation (1 3,000g for 20 min)then dissolved in 40 mm of Tris-HCl buffer (pH 7.5 at 25°C),containing 1 mM DTT, after which the solution was passedthrough a Sephadex G-25 column (1.6 x 15 cm) or a SephadexPD- 10 column, equilibrated with the same buffer.Enzyme Assay. Hyoscyamine 6fl-hydroxylase was assayed by

measuring the formation of 6f3-hydroxyhyoscyamine. The assaymixture contained (complete system) 50 mm of Tris-HCl buffer(pH 7.5 at 25°C), 0.4 mm of ferrous sulfate, 4 mM of sodiumascorbate, 1 mm of 2-oxoglutarate (neutralized with NaOH im-mediately before use), 0.2 mM of L-hyoscyamine hydrobromide,5% (v/v) acetone and the enzyme (0.2-0.5 mg of protein) in atotal volume of 1.0 ml. The reaction was started by adding theenzyme. The reaction was stopped by an addition of 0.1 ml ofapproximately 1.2 M Na2CO3-NaHCO3 buffer (pH 10); imme-diately, 1.0 ml of this alkaline reaction mixture was loaded on aExtrelut- 1 column, t-hen 6 ml of CHCl3 was passed through thecolumn. The CHC13 extract was evaporated to a dry residue at35°C which was dissolved in 0.1 ml of a mixture of 1,4-dioxaneand N,O-bis (trimethylsilyl)acetamide (4:1 v/v). 6fl-Hydroxy-hyoscyamine was quantified by GLC. Details of the GLC con-ditions are given in the legend to Figure 2 and elsewhere (1 1).

Protein was measured by the method of Lowry et al. (16).Crystalline BSA was the standard used.

Decarboxylation of 2-Oxoglutarate. The 2-oxoglutarate-de-grading activity was assayed by incubating the enzyme, hyoscy-

amine and the cofactors with [1-'4C]-2-oxoglutarate then trap-ping the labeled CO2 formed. The assay mixture was composedofenzyme (0.2-0.4 mg ofprotein), [ 1-'4C]-2-oxoglutarate (1 mm,0.055 ,uCi/ml), 5% (v/v) acetone, 50 mM Tris-HCI buffer, andhyoscyamine, ferrous sulfate and ascorbate in various concentra-tions; the total volume was 1.0 ml. The mixture was incubatedin stoppered test tubes (18 x 105 mm). A wire, to which wasattached a 1 x 4-cm rectangle of Whatman 3MM filter papermoistened with NCS reagent, was inserted through the stopperof the tube. After incubation at 30°C for 15 to 120 min, thereaction was stopped by injection of 200 ul of 25% (w/v) TCA.The tubes then were shaken at 30°C for 30 min to release the14C02 from the reaction mixture. The filter paper carrying the14C°2was transferred to a scintillation vial containing 10 ml ofUniver-Gell II, and the radioactivity determined in a LiquidScintillation Spectrometer (Packard C-2425).Enzyme Reaction in the Presence of N2. In this experiment,

the enzyme reaction took place under anaerobic conditions. Theassay mixture (0.9 ml) was placed in a stoppered vial (14 x 35mm), then nitrogen was bubbled through the solution for 10min. The gas was passed through the vial for another 30 min,then the enzyme (100 Al) was injected into the vial. The reactionwas stopped by an injection of 100 ul of carbonate buffer, thealkaloids being extracted immediately as described above.

Purification of the Product from the Reaction Mixture. Theassay mixture containing partially purified enzyme from about1 kg of cultured roots was incubated at 3O°C for 12 h and thereaction was stopped by adding 1 N HCI. After removing theprecipitates by centrifugation, the clear acidic solution was evap-orated to a dry residue which was dissolved in 0.1 N HCI, filteredand made alkaline (pH 10) with 28% NH40H. This solution wasloaded on a column containing Extrelute adsorbent, and CHC13was passed through the column. The CHC13 extract obtained wascondensed then applied to a preparative TLC plate coated withSilica gel 60 PF254. The plate was developed withCHCl3:ethanol:28% NH40H (85:15:4). Alkaloids were detectedwith a short UV wavelength, after which the silica-gel portionthat corresponded to the RF value of 6#-hydroxyhyoscyaminewas scraped from the plate.The silica gel collected was packed in a column which first was

eluted with CHC13 then with a mixture ofCHC13:methanol(3: 1).The CHCl3-methanol extract obtained was dried over anhydrousMgSO4 and evaporated to a dry residue which then was dissolvedin acetone. The acetone extract was made acidic with hydro-bromic acid, and the crystals (about 14 mg) that formed at 4°Cwere recrystallized from ethanol-ether.

Identification of the Product. Conditions for GC-MS (11) andTLC (25) have been described elsewhere.

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HYOSCYAMINE 6f-HYDROXYLASE IN ROOT CULTURES

RESULTS

Identification of the Reaction Product. When enzyme prepa-rations from Hyoscyamus niger root cultures were incubatedwith hyoscyamine, 2-oxoglutarate, Fe2" ions, and ascorbate, atime-dependent appearance of a new peak and a decrease inhyoscyamine were found on GLC chromatograms (Fig. 2). Theretention time of the new peak (Rt 6.18 min) corresponded tothat of authentic (-)-63-hydroxyhyoscyamine. GC-MS measure-ment gave a mass spectrum identical to that of the TMS-derivative of authentic (-)-6f3-hydroxyhyoscyamine. MS m/z(rel. int.) 449(2), 434(2), 333(4), 213(4), 212(20), 159(2), 145(2),122(3), 103(3), 96(14), 95(100), 94(66), 75(9), 73(25).Powdered samples of the purified reaction product, authentic

(-)-6f-hydroxyhyoscyamine and a mixture of both all melted at178 to 179°C. RF values for the TLC of both the authenticsamples and the product were identical; an RF of 0.49 with adeveloping solvent system of CHCl3:ethanol:28% NH40H(85:15:4) and an RF of 0.55 with a system of butanol:aceticacid:H20 (10:4:3). Their 'H NMR (200 M Hz, in CDC13) spectraalso were identical (see Fig. 1 for the numbering of protons); a7.20 to 7.42 (m, 5H) assigned to aromatic protons, 6 5.04 (broadt, J3,2 = J34 = 5.1 Hz, 1H) assigned to H-3, a 4.35 (dd, J6,7 = 7.3

A BE C

>, toj0 L.:>. . ...

0 2 4 6 0 2 4 6Retention Time ( min )

FIG. 2. Gas-liquid chromatograms of the reaction mixtures. Tropanealkaloids were extracted from incubated reaction mixtures and analyzedby GLC after converting the alkaloids to their TMS-derivatives as de-scribed in the text. Tricosane at a concentration of 0.5 mg/ml was addedas the internal standard before injection. Alkaloids were measured witha gas-liquid chromatograph, Shimadzu model GC-7A, equipped with acapillary column OV-101 (0.3 mm) x 25 m). The column temperaturewas 250°C and the carrier gas He at a flow rate of 0.92 ml/min. A splitratio of 54:1 and the detector FRD were used. A, The reaction was stoppedimmediately after the addition of enzyme, or the assay mixture wasincubated without enzyme for 1 h at 30°C; B, the assay mixture wasincubated with partially purified enzyme preparation (0.45 mg ofprotein)for 1 h at 30°C. Note the decrease in the hyoscyamine peak (Rt 4.70min) and the appearance of a new peak at Rt 6.18 min (1).

Hz, J6.5 = 2.4 Hz, 1H) assigned to H-6, 6 4.18 (X part of ABXpattern, 1H) assigned to H-2', a 3.81 (AB part of ABX pattern,2H) assigned to H2-3', 6 3.13 (quintet, J = 3.2 Hz, 1H) assignedto H-1, 6 3.00 (t-like, 1H) assigned to H-5, 6 2.50 (s, 3H) assignedto N-CH3, 6 1.95 to 2.65 (broad signal, 2H) assigned to protonsin hydroxy groups, 6 2.20 (ddd, Jgem = 15.9 Hz, J = 5.6 Hz, J =4.4 Hz, 1H) assigned to H-4, 6 2.09 (dt, Jgem = 15.4 Hz, J = 3.9Hz, 1H) assigned to H-2, 6 1.78 (dd, Jgem = 13.9 Hz, J = 7.1 Hz,1H) assigned to H-7, 6 1.68 to 1.50 (m, 2H) assigned to H-4 andH-7, 6 1.25 (broad d, Jgem = 15.4 Hz, I H) assigned to H-2.

Optical rotation values ([a]27) were -8.8 (c = 1.13, H20) forthe product and -7.4 (c = 0.87, H20) for the authentic alkaloid.The similar [a]D values found for the authentic sample and theproduct are evidence that the tropane-3,6-diol of the product hasthe 3S:6S absolute configuration reported for the alkamine ofauthentic (-)-6fl-hydroxyhyoscyamine (6). The preceding com-parisons establish the structure of the reaction product as (-)-6,B-hydroxyhyoscyamine.

Requirements of Hyoscyamine 6fl-Hydroxylase. In a typicalexperiment, fractionation of the crude extract with (NH4)2SO4(60-80% saturation) gave a 3.3-fold increase in the specificactivity of the enzyme and a 69% recovery. Partially purifiedenzyme preparations were used in the experiments reportedbelow.The conversion reaction had absolute requirements for hyos-

cyamine, 2-oxoglutarate, ferrous ions, and molecular oxygen;omission of any of these from the reaction mixture effectivelystopped the reaction (Table I; Fig. 3). Ascorbate stimulated theactivity 2.8-fold at a concentration of4 mm (Table I). Preliminaryexperiments with partially purified enzyme showed saturationcurves of a normal Michaelis-Menten type for hyoscyamine, 2-oxoglutarate and ferrous ions, with respective Km values of 17pM, 57 gm, and 9 gM. The enzyme uses only the L-isomer ofhyoscyamine as the alkaloid substrate; D-hyoscyamine was nearlyinactive (Fig. 4). Enzyme activity was proportional to the periodof incubation, 0 to 75 min, and to a concentration of protein of0 to 0.45 mg/ml (Figs 3 and 4).Acetone at concentrations of up to 10% (v/v) and catalase at

concentrations of 1 to 2 mg/ml stimulated enzyme activity, butthe extent varied with the purity of the preparation. Examples ofthe effects of acetone and catalase are shown in Table I. Becauseof its consistent stimulation of the partially purified enzyme, 5%acetone was included in all the reaction mixtures tested, exceptas shown in Table I.Hyoscyamine-Dependent Decarboxylation of 2-Oxoglutarate.

The decarboxylation of 2-oxoglutarate, a cofactor of hyoscy-amine 6,B-hydroxylase, was studied using carboxy-labeled 2-ox-oglutarate. When the enzyme was incubated with an assay mix-ture containing [1-'4C]-2-oxoglutarate, '4C02 was formed (Fig.5A). Even in the absence of hyoscyamine, a small amount of'4CO2 was formed during incubation, but the addition of hyos-cyamine greatly stimulated its evolution. The hyoscyamine-de-pendent yield of '4CO2 was calculated by subtracting the amountof '4CO2 formed in the reaction without hyoscyamine from thatin the complete reaction. This yield was plotted against theincubation period, along with that of the 613-hydroxyhyoscy-amine produced during incubation (Fig. SB). 6,3-Hydroxyhyos-cyamine and CO2 were produced in a 1:1- molar ratio whenhyoscyamine and 2-oxoglutarate were incubated under standardincubation conditions.

[1-'4C]-2-Oxoglutarate also was incubated under conditions inwhich the concentrations of hyoscyamine, ferrous ions and as-corbate were varied, the incubation time being set at 60 min.The yield of '4C02 plotted against the yield of 6,B-hydroxyhyos-cyamine, is shown in Figure 6. These results also indicate a 1:1stoichiometry between the decarboxylation of 2-oxoglutarate andthe hydroxylation of hyoscyamine (Y = 0.972X + 0.365, in

621




Table I. Cofactor Requirements ofHyoscyamine 6fl-HydroxylaseThe standard assay mixture was used with or without the additions shown. In experiment 1, 0.39 mg of

protein was incubated with each assay mixture for 1 h at 30C. In experiment 2, 0.30 mg of protein wasincubated with each assay mixture for 2 h at 30°C. Catalase was used at 2.1 mg/ml.

Experiment Omissions or Additions in Hyoscyamine 6(-Hydroxylase Activitythe Complete System

p Kat % ofcontrolNone 13.3 100- Hyoscyamine 0 0- 2-Oxoglutarate 0 0- Fe2+ 0 0-Ascorbate 4.8 36

2 None 11.1 100- Acetone 7.6 68+ Catalase 11.4 103- Acetone, + catalase 10.1 91

2

'a

E

IX

U

0)£si

x0L.

0 20 40 60

Reaction Time ( min )

1-

._2sE

x 40

0

80

FIG. 3. Formation of 6,#-hydroxyhyoscyamine from hyoscyamineafter different periods of incubation in the presence of air or N2. Theassay mixture had N2 gas bubbled through it for 10 min then wasincubated with partially purified enzyme preparation (0.35 mg ofprotein)in air (0) or in N2 (0). An assay mixture not subjected to N2 bubblingalso was incubated with the above enzyme preparation in air (A).

which Y is the yield of CO2 and X the yield of 6f,-hydroxyhyos-cyamine, both in nmol; r = 0.958, P < 0.001).Hyoscyamine 6#-Hydroxylase in Other Cultures. Various cul-

tures were tested for the presence of tropane alkaloids andhyoscyamine 6,B-hydroxylase activity (Table II). Tobacco rootcultures and shoot cultures of Duboisia (D. leichhardtii, D.myoporoides, and D. hopwoodii) produced no tropane alkaloids;there was no enzyme activity present in these cultures, exceptfor weak activity detected in a shoot culture of D. myoporoides.Some Datura root cultures (D. stramonium var inermis and varstramonium, and D. leichhardtii) and calluses of H. niger pro-duced only hyoscyamine; no enzyme activity was found.Root cultures ofAtropa belladonna, Duboisia leichhardtii, two

Datura species (D. fastuosa and D. innoxia), and five Hyoscy-amus species (H. niger, H. albus, H. gyorffi, H. pusillus, and H.muticus) produced both hyoscyamine and scopolamine, 6#-hydroxyhyoscyamine being present in several of them. Hydrox-ylase activity was found in the crude enzyme preparations fromall but one culture, regardless of the presence or absence of 6,B-hydroxyhyoscyamine in the root tissues; the one exception being

Protein ( mg )

FIG. 4. Formation of 6,B-hydroxyhyoscyamine from the L- or D-isomer ofhyoscyamine in incubations with different amounts ofpartiallypurified enzyme preparation. Various amounts of partially purified prep-aration were incubated at 30°C for 1 h in asay mixtures that eachcontained 200 nmol of L-hyoscyamine (0) or D-hyoscyamine (0).

the D. innoxia culture, which produced small amounts of 6,3-hydroxyhyoscyamine and scopolamine but had no detectableenzyme activity. Transfer of the root cultures to auxin-freemedium produced marked increases in the alkaloid contents andin the extractable hyoscyamine 6(#-hydroxylase activity in five ofthe seven scopolamine-producing cultures.The data given in Table II were analyzed statistically to deter-

mine whether there is any relation between hydroxylase activity(pKat/g fresh weight of cells) and the content or ratio of thealkaloids present. The correlation coefficient (r) for enzymeactivity and the combined alkaloid content of 6#-hydroxyhyos-cyamine plus scopolamine was 0.60 (n = 34, P< 0.001), whereasthe r value for enzyme activity and the alkaloid "ratio" of thecombined alkaloid content divided by the hyoscyamine contentwas 0.73 (n = 29, P < 0.001).




HYOSCYAMINE 63-HYDROXYLASE IN ROOT CULTURES

80

-

Ec 60

0

° 40-r

20

0'(80

0

E 60c

o 40-2

20

-

-6EC

0-.-u

80 EC0

60 -CEm

u

040 z

x0L

20 >K

%O0 20 40 60 80 100 1 20

Reaction Time ( min )

FIG. 5. Production of 14CO2 from [1-'4C]2-oxoglutarate (A) and thestoichiometry of the hyoscyamine-dependent production of "'CO2 andthe formation of 6,B-hydroxyhyoscyamine (B) after different periods ofincubation. A, Decarboxylation of 2-oxoglutarate was assayed by incu-bating the enzyme, cofactors and [1-"'C]2-oxoglutarate with (0) orwithout (0) 200 nmol of hyoscyamine. Then the "CO2 produced was

measured. B, Hyoscyamine-dependent production of 4C02 (A) was

calculated as described in the text. Formation of6,B-hydroxyhyoscyamine(A) was measured using GLC. See the text for details.

DISCUSSION

Properties of Hyoscyamine 6i-Hydroxylase. The identifica-tion of the reaction product, the co-factor requirements for thereaction, and the decarboxylation of2-oxoglutarate coupled withhydroxylation of a substrate firmly establish hyoscyamine 6,B-hydroxylase as a 2-oxoglutarate-dependent dioxygenase (EC1.14.1 1.-). Several 2-oxoglutarate-dependent dioxygenases areknown to oxidize substrates as diverse as proteins and pyrimi-dines (12), the best characterized being prolyl hydroxylase (2).Prolyl hydroxylase (20), flavanone 3-hydroxylase (7), and gibber-ellin 2fl-hydroxylase (13, 21) have been isolated from plant tissuesand characterized, to some extent, as this type of dioxygenase.On the basis of the data presented here and by analogy to

other well characterized 2-oxoglutarate-dependent dioxygenases,we conclude that hyoscyamine 6f,-hydroxylase catalyzes the re-action shown in Figure 7. The enzyme incorporates one atom ofmolecular oxygen into the tropane moiety at the C-6 of hyoscy-amine, forming 6,3-hydroxyhyoscyamine. This reaction is cou-pled with the incorporation of the other atom of molecularoxygen into 2-oxoglutarate, thereby decarboxylating the a-ketoacid to succinate and CO2. Formation of succinate during incu-bation ofenzyme preparations with the assay mixture was shownby GLC, and succinate was identified by GC-MS (T Hashimoto,Y Yamada, unpublished data).Hyoscyamine 6f,-hydroxylase oxidizes only the L-isomer of

0 1 0 20 30 40

6B-hydroxyhyoscyamine (n mol)

FIG. 6. Relation between the formation of 6(B-hydroxyhyoscyaminefrom hyoscyamine and the production ofCO2 from 2-oxoglutarate. Theformation of 6,B-hydroxyhyoscyamine is plotted against the formation of'4C02 during incubation with different concentrations of hyoscyamine,Fe2' and ascorbate. Three separate experiments with 0.2 to 0.4 mg ofprotein each were run for I h at 30°C.

hyoscyamine, D-hyoscyamine being nearly inactive. The weakhydroxylation that took place with the prepared D-hyoscyaminesolution (Fig. 4) may be caused by a small amount of contami-nating L-isomer in the D-hyoscyamine solution because it isdifficult to avoid some racemization of free D-hyoscyaminebefore the preparation of the hydrobromide salt (1). Plantssynthesize L-isomers of hyoscyamine and scopolamine (14). Al-though atropine, the racemic form of hyoscyamine, has beenisolated from some solanaceous species (3), usually racemizationoccurs during its isolation from plants (14).Hyoscyamine 6,8-Hydroxylase Activities in Various Cultures.

It has been reported that shoot cultures of Duboisia leichhardtii(24) and of a hybrid of D. leichhardtii and D. myoporoides (9)have the ability to synthesize scopolamine from hyoscyamine,but that their inability to supply hyoscyamine from roots pre-vents their accumulating the normal Duboisia alkaloids. Of thethree Duboisia species, D. myoporoides has the highest ratio ofscopolamine to hyoscyamine in its leaves. This may be why weakenzyme activity was detected only in the shoot culture of D.myoporoides, not in the shoot cultures of the other two species.Auxin, especially IBA, promotes the growth of cultured Hyos-

cyamus roots, mainly by stimulating lateral root induction, andgood growth of most Atropa, Datura, and Duboisia root culturescan only be maintained by supplementing the culture mediumwith 1 to 10 jLM IBA (1 1). The biosynthesis of scopolamine isinhibited as the concentration of IBA in the culture mediumincreases (1 1). When various root cultures, subcultured in IBA-containing medium, were transferred to, and cultured in, auxin-free medium, both the alkaloid contents and the activity ofhyoscyamine 6fl-hydroxylase increased in most of the cultures(Table II). Possibly, physiological or developmental changes pro-duced by IBA may suppress the enzyme activity that leads toalkaloid biosynthesis (e.g. that of hyoscyamine 6fl-hydroxylase)resulting in reduced amounts of alkaloids in root cultures cul-tured in IBA-containing medium.The general involvement of hyoscyamine 6,B-hydroxylase in

scopolamine biosynthesis shows that in plants hyoscyamine isconverted to 6,3-hydroxyhyoscyamine in one enzymic step.

623




Table II. Alkaloid Contents and Hyoscyamine 6fl-Hydroxylase Activity in Root, Shoot, and Callus CulturesMost root cultures were established as described previously (11). Tobacco root culture was obtained from

T. Endo. Hyoscyamus root cultures other than Hn 11 were subcultured in LS medium without auxin. Otherroot cultures were subcultured in LS medium with 1 or 10 Mm IBA. Prior to the experiments, parts of thecultures were transferred to auxin-free LS medium. Other culture conditions were the same as described in thetext. Part of these data has been reported (11). Duboisia shoot cultures and Hyoscyamus callus cultures wereestablished as described previously (24, 25), and cultured on LS agar medium, respectively, with 10 gM BAand with 10 Mm NAA and 5 Mm BA. Crude enzyme preparations were used in the enzyme assay.

Alkaloid ContentSpecies and Line Plant Hormone Hydroxylase ActivityhyoAt hyos-OH scop

JIM pKatlgfresh wt ofcells- % dry wtA. Root cultures

Nicotiana tabacum var Samsun

Datura stramonium var inermis

D. stramonium var stramonium

D. leichhardtii

D. innoxia

D. fastuosa

Atropa belladonna

Duboisia leichhardtiifl 5

S3232-2

K-4B

1753-4

Hyoscyamus nigerHnl 1

Hn 19H. albusH. gyorffiH. pusillusH. muticus

B. Shoot culturesDuboisia leichhardtiiD. myoporoidesD. hopwoodii

C. Callus culturesHyoscyamus nigerH15LH57

IBA 100

IBA 10

IBA 10

IBA 10

IBA 10

IBA 10

IBA 10

IBA 100

IBA 100

IBA 100

IBA 100

IBA 1000000

BA 10BA 10BA 10

NAA 10, BA SNAA 10, BA 5

a Each value is the mean of three measurements.amine; scop, scopolamine.

This finding argues against the proposed biosynthetic pathwayfor scopolamine (22) in which hyoscyamine is first dehydrogen-ated to 6,7-dehydrohyoscyamine. If 6,7-dehydrohyoscyamine isa precursor of scopolamine, it shoud be located between 6fl-hydroxyhyoscyamine and scopolamine (14, 19).Two configurations of the tropane-3,6-diol are present in the

Solanaceae. The alkamine of 6,3-hydroxyhyoscyamine and vale-roidine, an ester with isovaleric acid isolated from Datura andDuboisia, has the (-)-form. The 3S:6S absolute configurationhas been assigned to this alkaloid (6, 18). The hyoscyamine 6,B-hydroxylase described by us hydroxylates the tropane-3a-ol ofhyoscyamine to the 3S:6S tropane-3,6-diol. The (+)-enantiomer

00000000002.52.82.97.5

4.517.45.9

22.14.2

11.14.84.0

22.633.115.03.5

17.92.914.6

01.40

00

000.1090.2010.2570.4230.1880.1840.2320.3820.1230.1080.1280.270

0.0940.1280.1690.1040.0810.3120.1100.164

0.0240.0250.0160.4500.2760.1000.084

000

000000000.0240.0360.0390.0190.0270.065

0.0370.0530.1210.0330.0170.0660.0350.037

0000.0290.03800

000

000000000.0410.05 10.1070.1010.0140.016

0.0970.1780.2850.3150.0460.2740.2070.354

0.1130.2120.1730.0520.3010.0230.163

000

0.001 0 00.001 0 0

b hyos, hyoscyamine; hyos-OH, 6,B-hydroxyhyoscy-

is the alkamine of the mono- and di-tigloyltropane diols foundin Datura and has the 3R:6R configuration (6, 18). Major et al.(17) in a preliminary report showed that the Cyt P450 of rat livermicrosomes catalyzes the conversion of 3a-tigloyloxytropane to3a-tigloyloxytropane-6,7#-diol (meteloidine). In plants, metelo-idine is assumed to be formed by hydroxylation of 3a-tigloylox-ytropane via 3a-tigloyloxytropane-7#-ol, a 3R:6R tropane-3,6-diol ester with tiglic acid (14). Therefore, in terms of the stereo-specificity of hydroxyl group introduction, Major's rat livermicrosomes might catalyze the hydroxylation of the tropane-3a-ol entirely differently than the hyoscyamine 6f-hydroxylase re-ported here. It would be interesting to know whether different




HYOSCYAMINE 6,3-HYDROXYLASE IN ROOT CULTURES

N -cMe

O.,C-CHIllI0 CH20H

l-Hyoscyamine

2e < 2-oxoglutaric Acid + 02Fe2

Ascorbate

A

CO2

N- Me

HO

°\'C-CH-0O CH20H

6 B-Hydroxyhyoscyami ne

FIG. 7. Reaction of hyoscyamine 6fl-hydroxylase. See text for details.

groups ofenzymes (2-oxoglutarate-dependent dioxygenase or CytP450 monooxygenase) function in the hydroxylation of differentcarbons (C-6 or C-7) in the tropane moiety of alkaloids.

Acknowledgments-We thank Y. Yukimune for his help in preparing the D-hyoscyamine; Dr. K. Inoue, Faculty of Pharmaceutical Sciences, Kyoto University,for measuring the 'H-NMR and for the assignment of the signals; K. Irie, Facultyof Agriculture, Kyoto University, for measuring the [a]D; and P. Yamada forcorrecting our English.

LITERATURE CITED

1. BARROWCLIFF M, F TUTIN 1909 The configuration of tropine and 4'-tropine,and the resolution of atropine. J Chem Soc 95: 1966-1977

2. CARDINALE GJ, S UDENFRIEND 1974 Prolyl hydroxylase. Adv Enzymol 41:245-300

3. EVANS WC 1979 Tropane alkaloids of the Solanaceae. In JG Hawkes, RNLester, AD Skelding, eds, The Biology and Taxonomy of the Solanaceae.Academic Press, London, pp 241-254

4. EVANS WC, KPA RAMSEY 1983 Alkaloids of the Solanaceae tribe Anthocerci-deae. Phytochemistry 22: 2219-2225

5. FODOR G, A ROMEIKE, G JANZSO, I KOCZOR 1959 Epoxidation experiment invitro with dehydrohyoscyamine and related compounds. Tetrahedron Lett7: 19-23

6. FODOR G, F SOTI 1964 Correlation of valeroidine with S(-)methoxysuccinicacid and of mono- and ditigloyltropane-3,6-diol with its R(+)antimer. Tet-rahedron Lett 29: 1917-1921

7. FORKMANN G, W HELLER, H GRISEBACH 1980 Anthocyanin biosynthesis inflowers of Matthiola incana: Flavanone 3- and flavonoid 3'-hydroxylases. ZNaturforsch 35c: 691-695

8. GAMBORG OL, RA MILLER, K OJIMA 1968 Nutrient requirements of suspen-sion cultures of soybean root cells. Exp Cell Res 50: 151-158

9. GRIFFIN WJ 1979 Organization and metabolism of exogeneous hyoscyaminein tissue cultures of a Duboisia hybrid. Naturwissenchaften 66: 58

10. HASHIMOTo T, Y YAMADA 1983 Scopolamine production in suspension cul-tures and redifferentiated roots of Hyoscyamus niger. Planta Med 47: 195-199

11. HASHIMoTo T, Y YUKIMUNE, Y YAMADA 1986 Tropane alkaloid productionin Hyoscyamus root cultures. J Plant Physiol. In press

12. HAYAISHI 0, M NOZAKI, MT ABBOTT 1975 Oxygenases: Dioxygenases. In PDBoyer, ed, The Enzymes, Ed 3, Vol 12. Academic Press, New York, pp 1 19-189

13. HOAD GV, J MACMILLAN, VA SMITH, VM SPONSEL, DS TAYLOR 1982 Gib-berellin 2 -hydroxylases and biological activity of 2 -alkylgibberellins. In PFWareing, ed, Plant Growth Substances 1982. Academic Press, London, pp

91-10014. LEETE E 1979 Biosynthesis and metabolism of the tropane alkaloids. Planta

Med 36: 97-11215. LINSMAIER EM, F SKOOG 1967 Organic growth factor requirements of tissue

cultures. Physiol Plant 51: 100-12716. LOWRY OH, NH ROSEBROUGH, AL FARR, Rl RANDALL 1951 Protein meas-

urement with the Folin phenol reagent. J Biol Chem 193: 265-27517. MAJOR EWT, II DAVIES, JG WOOLLEY 1978 The hydroxylation of tropane

alkaloids: J Pharm Pharmacol 30: 81P18. ROMEIKE A 1978 Tropane alkaloids-occurrence and systematic importance

in angiosperms. Bot Notiser 131: 85-9619. ROMEIKE A, G FORDOR 1960 The biogenesis ofhyoscine in Datura stramonium

L. Tetrahedron Lett 22: 1-420. SADAVA D, MJ CHRISPEELS 1971 Hydroxyproline biosynthesis in plant cells:

Peptidyl proline hydroxylase from carrot disks. Biochim Biophys Acta 227:278-287

21. SMITH VA, J MACMILLAN 1984 Purification and partial characterization of a

gibberellin 2 -hydroxylase from Phaseolus vulgaris. J Plant Growth Regul 2:251-264

22. WALLER GR, EK NOWACKI 1978 Alkaloid Biology and Metabolism in Plants.Plenum Press, New York, p 71

23. WERNER G, K MILTENBERGER 1960 Zur Trennung der Optischen Antipodenvon Homatropine und Atropine; Synthese von L(+)- und D(-)-Homatropin-sulfat. Liebigs Ann Chem 631: 163-168

24. YAMADA Y, T ENDO 1984 Tropane alkaloid production in cultured cells ofDuboisia leichhardtii. Plant Cell Rep 3: 186-188

25. YAMADA Y, T HASHIMOTO 1982 Production of tropane alkaloids in culturedcells of Hyoscyamus niger. Plant Cell Rep 1: 101-103

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