Monoterpene synthases from Conocephalum conicum
368
reagent with lysozyme as standard# was diluted to 199 the extract was concd for radio!GLC as before "the
ml with extraction bu}er containing 09 mM MgCl in column was programmed from 049> "4 min iso!
1
−
0
a 0[6 ml Eppendorf tube[ When it was necessary to thermal# to 129> at 4> min #[
modify these conditions\ for example to determine the
Enzyme characterization[ To determine the opti!
response to pH or to di}erent cations\ the samples mum pH and bu}er type for monoterpene synthase
were dialysed to the appropriate conditions[ The reac! activity\ assays were conducted at half pH unit inter!
2
tion was initiated by the addition of 4 mM ð0! HŁger! vals in Tris "pH 5[4Ð7[4#\ HEPES "pH 5[4Ð7[4#\ phos!
anyl diphosphate "9[14 mCi# followed by gentle mixing phate "pH 6[9 and 6[4#\ Bis!Tris propane "pH 6[4Ð
and incubation for 0 hr at 21>[ After incubation\ 0 09[9# and AMPSO "pH 6[9Ð7[4#\ all at 29 mM con!
ml hexane was added and the reaction mixture was taining 09 mM b!mercaptoethanol\ 09 mM MgCl and
1
vigorously mixed and\ following centrifugation to sep! 09) glycerol[ For studies of divalent cation require!
arate phases\ 9[8 ml of the hexane layer was trans! ment\ preparations were desalted into 19 mM HEPES
ferred to a new Eppendorf tube containing 099 mg bu}er "pH 6[9# containing 09 mM b!mercaptoethanol\
silica gel ðSilicAR 59A "Millinckrodt#Ł\ followed by 09 mM sodium ascorbate\ 09 mM Na S O and 09)
1
1
4
mixing and centrifugation to pellet the matrix and glycerol by passage through an Econo!Pac 09 DG
a}ord the hydrocarbon fraction\ free of oxygenated column "Bio!Rad#[
metabolites[ Ether extraction of the silica gel "0 ml
To estimate the native molecular mass of the sabi!
Et O# provided the oxygenated monoterpenes[ A 9[4 nene synthase\ the partially puri_ed\ concd enzyme
1
ml portion of the monoterpene hydrocarbon fraction prepn was subjected to size!exclusion chro!
and of the monoterpene alcohol fraction was analysed matography on a calibrated Sephacryl S!199 column
by liquid scintillation counting in 09 ml of a cocktail "0[5 cm×89 cm\ Pharmacia#\ developed with extrac!
consisting of 9[3) Omni~uor "DuPont:New England tion bu}er at pH 6[9 containing 099 mM KCl at a
2
−0
Nuclear# dissolved in 29) EtOH in toluene " H ~ow!rate of 9[4 ml min [ The Km value for geranyl
e.ciency\ ¼31)#[ The assay at 4 mM substrate was diphosphate with the sabinene synthase was deter!
linear with respect to protein concn up to at least 19 mined by computer assisted Lineweaver!Burk plotting
−
0
mg ml for a 0 hr incubation period\ and all assays of the reaction rates obtained over a substrate con!
were conducted under these linear conditions[ Boiled centration range of 9[4 to 04 mM[
2
controls evidenced negligible activity with ð0! HŁger!
anyl diphosphate as substrate[
Acknowled`ements*We thank Professor Hans
The identity and purity of the biosynthetic products Becker for support and encouragement\ Alfred Koepp
were determined by radio!GLC ð29Ł[ To prepare and John Crock for technical assistance\ and Joyce
su.cient product\ the assay was scaled!up using 0 ml Tamura!Brown for typing the manuscript[ K[!P[A[
of enzyme prepn and the incubation time was thanks the Deutsche Forschungsgemeinschaft for
extended to 2 hr[ The monoterpene hydrocarbon frac! _nancial support[ This work was supported by
tion\ obtained after chromatography of the hexane National Institutes of Health Grant GM!20243 and
extract over silica gel\ was diluted with authentic stan! by Project 9157 from the Washington State University
dards\ concd and analysed[ The conditions for analy! Agricultural Research Center[
sis on the 2 mm o[d[×3 m AT0999 column "04)
polyethylene glycol ester on Gas!Chrom Q# were 099>
REFERENCES
isothermal "4 min# then programmed to a _nal temp[
−
0
−0
of 199> at 4> min \ with helium "23 ml min # as
carrier\ injector at 129>\ and thermal conductivity
detector at 149> with current of 049 mA[ The identity
of the enzymatically!generated monoterpene ole_n
0[ Asakawa\ Y[\ in Pro`ress in the Chemistry of
Or`anic Natural Products\ Vol[ 31\ ed[ W[ Hertz\
G[ W[ Kirby\ R[ E[ Moore\ W[ Steglich and Ch[
Tamm[ Springer\ Wien\ 0871\ p[ 0[
"
"−#!sabinene# was veri_ed by comparison of the Rt
1[ Asakawa\ Y[\ in Biolo`ical Natural Products!
Detection\ Isolation and Structural Determination\
ed[ S[ M[ Colegate and R[ J[ Molyneux[ CRC
Press\ Boca Raton\ FL\ 0882\ p[ 208[
2[ Asakawa\ Y[\ in Pro`ress in the Chemistry of
Or`anic Natural Products\ Vol[ 54\ ed[ W[ Hertz\
G[ W[ Kirby\ R[ E[ Moore\ W[ Steglich and Ch[
Tamm[ Springer\ Wien\ 0884\ p[ 0[
3[ Asakawa\ Y[\ in Bryophytes\ their Chemistry and
Chemical Taxonomy[ Proceedin`s of the Phy!
tochemical Society of Europe\ ed[ H[ D[ Zinsmeis!
ter and R[ Mues[ Clarendon Press\ Oxford\ 0889\
p[ 258[
4[ Zinsmeister\ H[ D[\ Becker\ H[ and Eicher\ T[\
An`ewante Chemie International Edition\ 0880\ 29\
029[
to the authentic standard[ In the assay for bornyl
diphosphate\ the bulk of the ð0! HŁgeranyl diphos!
2
phate remaining after enzyme incubation was
removed by addition of 099 ml of 9[0 M HCl to sol!
volyse the allylic substrate "primarily to linalool\ ger!
aniol and nerol#\ followed by hexane extraction[ The
pH of the residual aqueous layer was adjusted to 5[4
with dilute NaOH\ and the mixt[ was incubated with
0
unit each of apyrase and acid phosphatase to hydro!
lyse phosphate esters of borneol[ Following this enzy!
matic hydrolysis step and hexane extraction\ the
extract was diluted with authentic "¦#!borneol\ concd
and puri_ed by TLC "silica gel\ hexaneÐEtOAc\ 6]2#[
The band corresponding to borneol "R ¼ 9[38# was
removed\ the silica gel was extracted with Et O\ and
f
1