Purification and characterization of acetyl coenzyme A: 10- hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxus chinensis

Article abstract of DOI:10.1016/S0031-9422(98)00674-8

An O-acetyltransferase that catalyzes the regiospecific acetylation of a range of taxanes possessing an unsubstituted 10-hydroxyl group was detected and purified to apparent electrophoretic homogeneity from a cytosolic fraction of Taxus chinensis cell cultures. The purification involved negative calcium phosphate adsorption, sephadex desalting, DEAE, AcA44 chromatography, HighQ, CHT II, HiTrap Blue, Phenylsepharose and Mimetic Green purification steps. The purified acetyltransferase was found to be a monomeric protein of 71 ± 1.5 kDa that is highly regio- and stereospecific towards the 10β- hydroxyl group of the taxane molecule and is also active towards 10- desacetylbaccatine III. The acetyltransferase reaction had a pH optimum of 9.0 with halfmaximal activities at pH 6.8 and 10.8, respectively. The temperature optimum was at 35°C and the isoelectric point at 5.6. The apparent K(m) values for 10-desacetyltaxuyunnanine C and acetyl CoA were 23 and 61 μM, respectively. The turnover rate for the enzyme using both substrates was 0.2 mol mol^-1 of enzyme. The kinetic optimum was determined to be K(cat)/K(m) = 8.7 s^-1 L M^-1.

Full text of DOI:10.1016/S0031-9422(98)00674-8

\
PERGAMON
Phytochemistry 49 "0888# 652Ð663
Special publicationꢀ
Puri_cation and characterization of acetyl coenzyme A]
09!hydroxytaxane O!acetyltransferase from cell suspension
cultures of Taxus chinensis
Birgitta Menhard\ Meinhart H[ Zenk$
Lehrstuhl fur Pharmazeutische Biologie\ Universitat Munchen\ Karlstrasse 18\ D!79222 Munchen\ Germany
Ã
Ã
Ã
Ã
Received 17 July 0887
Abstract
An O!acetyltransferase that catalyzes the regiospeci_c acetylation of a range of taxanes possessing an unsubstituted 09!hydroxyl
group was detected and puri_ed to apparent electrophoretic homogeneity from a cytosolic fraction of Taxus chinensis cell cultures[
The puri_cation involved negative calcium phosphate adsorption\ sephadex desalting\ DEAE\ AcA33 chromatography\ HighQ\
CHT II\ HiTrap Blue\ Phenylsepharose and Mimetic Green puri_cation steps[ The puri_ed acetyltransferase was found to be a
monomeric protein of 6020[4 kDa that is highly regio! and stereospeci_c towards the 09b!hydroxyl group of the taxane molecule
and is also active towards 09!desacetylbaccatine III[ The acetyltransferase reaction had a pH optimum of 8[9 with halfmaximal
activities at pH 5[7 and 09[7\ respectively[ The temperature optimum was at 24>C and the isoelectric point at 4[5[ The apparent Km
values for 09!desacetyltaxuyunnanine C and acetyl CoA were 12 and 50 mM\ respectively[ The turnover rate for the enzyme using
both substrates was 9[1 mol mol−0 of enzyme[ The kinetic optimum was determined to be Kcat:Kmꢁ7[6 s−0 L M−0[ Þ 0888 Elsevier
Science Ltd[ All rights reserved[
Keywords] Taxus chinensis^ O!acetyltransferase^ Cell culture^ 09!Desacetyltaxuyunnanine C^ 09!Desacetylbaccatine III
0[ Introduction
the genus Taxus through molecular genetic approaches[
At least two strategies can be considered to accomplish
this^ either to deregulate the biosynthesis of this precursor
in di}erentiated plants and cell cultures or to clone and
overexpress rate limiting enzymes in Taxus species to
boost taxane production[
Taxolþ "0# and Taxotereþ "1# are at present successful
anticancer drugs of steadily increasing medical usage
"Wani\ Taylor\ Wall\ Coggon\ + McPhail\ 0860#[ The
original limited supply of Taxolþ "0# from natural
resources "Wani et al[\ 0860# was overcome by a sem!
isynthetic process allowing for the production of both
Taxolþ "0# and Taxotereþ "1# from 09!desacetylbaccatine
III "09!DAB III# "2\ Figs 0 and 1#\ which is available in
su.cient quantities "up to 9[1) dry weight# from needles
Studies on the biosynthesis of 0 and 2 can be divided
into three di}erent chapters[ The _rst is the origin of the
terpenoid precursor for the biosynthesis of the taxane
carbon skeleton\ which has been proven to follow the
0!desoxyxylulose and not the mevalonate pathway
"Eisenreich\ Menhard\ Hylands\ Zenk\ + Bacher\ 0885#[
The second point is the formation of the taxa!3"4#\00"01#!
diene ring system starting from geranylgeranyl!
pyrophosphate as substrate\ which was most elegantly
shown recently "Koepp\ Hezari\ Zajicek\ Stofer!Vogel\ +
La Fever\ 0884^ Hezari\ Lewis\ + Croteau\ 0884^ Wildung
+ Croteau\ 0885^ Hezari\ Ketchum\ Gibson\ + Croteau\
0886#[ Third\ the substitution of the tricyclic terpenoid
by eight oxygenation reactions\ two acetylations\ one
benzoylation and one sidechain addition at C!02 "Floss
+ Mocek\ 0884^ Hezari + Croteau\ 0886#[
of the European yew Taxus baccata L[ "Senilh\ Gueritte!
ꢂ
ꢂ
ꢂ
ꢂ
Voegelein\ Guenard\ Colin\ + Potier\ 0873^ Gueritte!
Voegelein\ Senilh\ David\ + Guenard\ 0875#[ In order to
ꢂ
ꢂ
secure a long!term supply of both anticancer drugs\ the
biosynthesis of this important precursor clearly needs to
be studied with the aim of increasing the amount of 2 in
ꢀ Papers with this heading have received accelerated publication\ due
to their particular signi_cance in the _eld of phytochemistry[
$ Corresponding author[ Tel[] ¦38!78!4891!133^ fax] ¦38!78!4891!
500^ e!mail] zenkÝlrz[uni!muenchen[de
9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S9920!8311 "87#99563!7

653
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Fig[ 0[ Structures of naturally occurring taxanes[
One of the late steps in the biosynthesis and semi!
the speci_city of the 09!acetylation reaction\ we set out
to purify and characterize this enzyme activity using 4 as
a substrate[ It should be clearly noted that taxuyunnanine
C "4# possesses a 03!hydroxyl group and no substitution
at C!02[ This is at variance with 0 and 2\ which possess
the C!02 oxygen function and are unsubstituted at C!03[
In this study we describe the isolation and charac!
terization of an acetyl coenzyme a]acetyltransferase from
a T[ chinensis cell culture that is highly regio! and stereo!
speci_c for the 09b!hydroxyl group of 6 but surprisingly
also accepts 09!DAB III "2# and congeners as substrates[
synthesis of Taxolþ "0# is the acetylation at the C!09
position of 09!DAB III "2# to yield baccatine III "3#[ This
compound is assumed to be the immediate precursor of
0 "Floss + Mocek\ 0884#[ This acetylation reaction has
recently been described in insu.cient form using acetyl
CoA as a cosubstrate and a crude enzyme mixture from
needles of T[ baccata and lacking controls "Zocher et al[\
0885#[ A speci_c enzymic 09!acetylation of 2 would be of
considerable interest\ because it potentially circumvents
the introduction of a protecting group at C!6 in the
chemical acetylation reaction during the synthesis of 0
"Gueritte!Voegelein et al[\ 0875#[
ꢂ
During our investigation on the origin of the terpenoid
precursor of the taxane carbon skeleton "Eisenreich et
al[\ 0885# we used a cell culture of T[ chinensis "Pilger#
Rehd[\ which produced up to 69 mg l−0 of 1a\4a\09b\03b!
tetraacetoxy!3"19#\00!taxadiene "taxuyunnanine C "4##[
This yield could be increased up to consistent levels of 049
mg l−0 by medium optimization "Menhard\ Eisenreich\
Hylands\ Bacher\ + Zenk\ 0887#[ In order to investigate
1[ Results
1[0[ Preparation of substrates
Readily available 4 isolated from the optimized T[ chi!
nensis cell culture "Eisenreich et al[\ 0885# was used for
the preparation of select desacetylation products[ Figure

B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
654
Fig[ 1[ Compounds generated by desacetylation of taxuyunnanine C "4#[

655
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Fig[ 2[ Kinetics of the desacetylation of taxuyunnanine C "4# by incubation with "a# 9[0 M NaOH and "b# 0 M NaOH[ This mixture was incubated
at 29>C[ The progress of the reaction was monitored by analyzing aliquots by HPLC[ Structures of the compounds in question are shown in Fig[ 1[
2a shows the kinetics of the desacetylation reaction using
9[0 M NaOH at 29>C[ Under these conditions\ 6 was
synthesized in 59) yield[ The compound was isolated
using semipreparative HPLC and its structure was veri!
_ed by MS and NMR[ 6 was the main substrate used
for the detection and isolation of the acetyltransferase[
Desacetyltaxuyunnanine derivatives containing two\ one
or no acetyl group\ were isolated from hydrolysis experi!
ments using 4 but with more severe conditions such as 0
N NaOH at 29>C "Fig[ 2b#[

B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
656
Fig[ 3[ Reaction sequence catalyzed by acetyl CoA]09!hydroxytaxane O!acetyltransferase[
1[1[ Enzyme assay and enzyme detection
apparent homogeneity as determined by SDS!PAGE[
The puri_cation procedure is summarized in Table 0[
The enzyme in question was thought to be an acetyl
CoA!dependent acetyltransferase\ catalyzing the acetyl
transfer from the thiolester onto the only free hydroxyl
group in position 09 of 6 "Fig[ 3#[ A radioactive assay
was chosen for a reliable quantitative and sensitive
testing[
1[2[ pH optimum\ temperature optimum and isoelectric
point
The pH optimum of the enzyme was measured under
standard assay conditions[ The enzyme showed a pH
optimum at 8[9 with half maximal activities at 5[7 and
09[7\ respectively[ The optimal temperature for the re!
action was at 24>C[ Half maximal activities were at 05
and 32>C\ respectively[
The isoelectric point was determined by chro!
matofocusing on a polybu}er exchanger "Mono P HR#
column to be at pH 4[5[
The molecular mass of the acetyltransferase was deter!
mined by two methods[ Using a previously calibrated
Bioselect SEC 149!4 FPLC column\ the enzyme activity
was eluted at a volume corresponding to a protein of
approximately 61 kDa[ Under denaturing conditions the
puri_ed enzyme showed "after Ag¦ staining# a protein
band at 69[8 kDa[ The results indicate that the acetyl!
transferase is a monomeric protein with a molecular mass
of 6020[4 kDa[
No enzyme activity could be detected in crude extracts
of T[ chinensis suspension cells\ which was most likely
due to an excessive amount of phenols in the crude
extract[ This extract was\ therefore\ treated with calcium
phosphate gel\ subjected to gel _ltration and then\ with
the resulting protein fraction still showing no enzyme
activity\ passed through a DEAE!Sephacel column[ The
brownish colored protein fraction that was not absorbed
again did not show any enzyme activity but 73) of
protein from the crude cell extract had been removed by
the three previous steps[ The protein fraction eluting from
the DEAE column was applied to a column containing
Ultrogel AcA33[ A total of 59 fractions "Fig[ 4a# were
collected and for the _rst time enzyme activity could
be detected[ If this protein fraction was assayed in the
presence of 6 and ð0!03CŁacetyl CoA\ the product of the
reaction was clearly 4 as shown by TLC and HPLC
analysis[
When\
however\
1\4\09\03!desacetyl!
1[3[ Product identi_cation
taxuyunnanine C "00# was used as a substrate\ the sole
product formed was also 4[ This clearly showed that in
the semipuri_ed extract either one unspeci_c enzyme that
acetylated all four possible hydroxyl groups of the taxane
used as substrates was present or that the extract con!
tained a mixture of at least four di}erent and regiospeci_c
acetyltransferases that sequentially acetylated the com!
pound 00 in a position!speci_c matter[ To address this
question\ 6 was used as a sole substrate furtheron for the
subsequent puri_cation of the target enzyme by anion
exchange chromatography "HighQ#\ hydroxylapatite\
HiTrap Blue\ Phenylsepharose and Mimetic Green as
summarized in Fig[ 4bÐe[ These chromatographic steps
resulted in a 179!fold increase in speci_c activity with a
9[0) recovery[ The enzyme was obviously puri_ed to
The puri_ed acetyltransferase catalyzed the transfer of
the acetyl moiety of acetyl CoA to 6[ In order to charac!
terize the reaction product of this transfer\ ð0\1!02C1Łacetyl
CoA was incubated with unlabelled 6[ After incubation
for 0 h at 24>C\ the product of the reaction was extracted
with ether and puri_ed _rst by TLC and subsequently by
HPLC[ A single compound was isolated that had the
same Rf and Rt value as well as the same UV spectrum
as authentic 4[ The compound was subjected to MS
analysis and showed a clear and unequivocal ðM¦0Ł¦
peak of 496 "CI mode^ Fig[ 5b# in contrast to the 4
reference showing an ðM¦0Ł¦ at 494[ The latter spectrum
is shown in Fig[ 5a[ Clearly\ only one ð0\1!02C1Łacetyl
group was transferred to 6\ and since the substrate taxane

657
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Fig[ 4[ Puri_cation of 09!hydroxytaxane O!acetyltransferase[ "a# Elution pro_le of an AcA 33 column[ "b# Elution pro_le of a HighQ column[ "c#
Elution pro_le of a CHT II column[ "d# Elution pro_le of a HiTrap Blue column[ "e# Elution pro_le of a phenylsepharose column[ Collected fractions
were assayed with the standard enzyme assay[ The bars indicate the combined fractions\ which were used for subsequent enzyme puri_cation[
was unequivocally characterized as 6\ the puri_ed enzyme
must have catalyzed the stoichiometric transfer of one
acetyl group from acetyl CoA to the 09!position of 0 mol
of the acceptor molecule[
1[4[ Substrate speci_city
A crucial test was whether this acetyltransferase cat!
alyzed the regiospeci_c acetylation only at the 09!position
of desacetyltaxuyunnanine C derivatives or whether this
enzyme catalyzed unspeci_cally all four or select
hydroxyl!groups in 1\4\09\03!desacetyltaxuyunnanine C
"00#[ Incubation of the latter substrate with the pure
enzyme and ð1!03CŁacetyl CoA showed that only one
hydroxyl group was acetylated\ which ruled out that the
target enzyme was not selective and acetylated all four or
The Km value of acetyl CoA was calculated from the
LineweaverÐBurk plot to be 50 mM\ while the Km value
for 6 was determined to be 12 mM for this enzyme[ The
turnover with these substrates was 9[1 kat mol−0 at opti!
mal conditions using the homogenous enzyme[ The
kinetic optimum "kcat:Km# for the reaction was calculated
to be 7[6 s−0 L M−0
[

B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
658
Table 0
Puri_cation of acetyl CoA]09!hydroxytaxane O!acetyltransferase from Taxus chinensis cell cultures
Puri_cation step
Total protein
"mg#
Total activity
"pkat#
Speci_c activity
Recovery
")#
Puri_cation!
"fold#
"pkat mg−0
#
Raw extract
Ca2"PO3#1
261
083
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Sephadex G14
DEAE
67
48
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
AcA33
HighQ
CHT II
HiTrap Blue
Phenylsepharose
Mimetic Green
14[6
7[7
9[62
9[94
9[990
9[9990
613
685
467
057
5[2
9[6
17
099
009
79
12
9[76
9[0
0
89[4
681
2259
5299
6999
2[1
1b
019
114
149
several free hydroxyl groups in that substrate molecule[
The substrates 09\03! "7#\ 1\09\03! "09#\ as well as 4\09\03!
desacetyltaxuyunnanine C "8#\ were singly acetylated as
determined by radioTLC and radioHPLC on comigration
with authentic standards[ A crucial experiment was
whether 09!DAB III "2# can act as an acetyl acceptor for
the transferase[ Indeed 2 was also singly acetylated[ Incu!
bation of 2 in the presence of enzyme and ð0\1!02C1Łacetyl
CoA gave a product that was indistinguishable in its chro!
matographic behavior "TLC\ HPLC# from baccatine III
"3#[ Mass spectral analysis of the labelled product yielded
in the CI!mode a clear ðM¦0Ł¦ peak at 478 m:z "Fig[ 6b#
spectrometric proof of its identity as baccatine III was
presented[ This is insu.cient evidence to base the assertion
that a regiospeci_c acetyltransferase had been isolated[
The second acetyltransferase is speci_c for the 4!
hydroxyl group of the taxane nucleus and has been
reported only in preliminary form "Hezari + Croteau\
0886#[ This enzyme was partially puri_ed and shown to
be a 49 kDa monomeric protein with an isoelectric point
of ½3[6[ Both characteristics as well as the regios!
peci_city of the enzyme are clearly di}erent from the
enzyme discovered and characterized in this study[
There is no doubt that in T[ chinensis cell cultures there
are di}erent acetyltransferases with di}erent regios!
peci_city towards the di}erent hydroxyl groups of the
taxane ring system\ as represented by the substrate 00[
The _rst fraction "AcA33 eluate# showing ace!
tyltransferase activity in our puri_cation scheme Table 0
acetylated in the presence of acetyl CoA all four hydroxyl
groups of the taxane\ yielding taxuyunnanine C "4# as a
reaction product[ In contrast\ the puri_ed enzyme that
was isolated using 6 as the only substrate\ is absolutely
regiospeci_c for the 09!position[ Furthermore\ not only
is 09!desacetyltaxuyunnanine C "6# monoacetylated\ but
other partly desacetylated taxuyunnanine C derivatives
are also acetylated only at the 09!position Fig[ 3[ More!
over\ 09!DAB III "2# as well as 08!hydroxy!09!DAB III
is only acetylated at this 09!hydroxyl position[
in comparison to the reference compound "476 ðM¦0Ł¦0
^
Fig[ 6a#[ Interestingly\ the 09!epi!09!DAB III that has the
a!con_guration at the 09!hydroxyl group is not at all ace!
tylated\ indicating that the enzyme is not only regio!\ but
also stereoselective[ The _nding that 1\4!desacetyl!09\03!
desacetoxy!taxuyunnanine C\ which lacks the hydroxyl
group recognized by the acetyl transferase\ is inactive as a
substrate underscores the regioselectivity of the enzyme[
The results of the substrate speci_city are shown in Table
1[ The new enzyme is named acetyl coenzyme A]09!hyd!
roxytaxane O!acetyltransferase and catalyzes the reaction
shown in Fig[ 3[ It is a soluble enzyme and membranous
fractions are devoid of this enzyme activity[ This enzyme
was found to occur in cell cultures of a number of Taxaceae
including T[ baccata\ T[ media and Torreya taxifolia[
We have demonstrated for this _rst time a regio! and
stereoselective acetyltransferase that selectively acetylates
the 09 b!hydroxyl position in a number of taxanes[ This
shows clearly that functionalization of the diterpenoid
ring system requires highly speci_c enzymes[
2[ Discussion
To this point only two acetyltransferases involved in
the biosynthesis of naturally!occurring taxanes had been
reported in the literature and neither of these enzymes had
been extensively puri_ed or characterized[ One transferase
was claimed to occur in roots of T[ baccata "Zocher et
al[\ 0885#[ This enzyme was reported to regiospeci_cally
acetylate 2 at the 09!position with acetyl CoA as acetyl
donor[ The reaction product\ however\ was characterized
only by TLC\ HPLC and radioscanning and no rigorous
3[ Experimental
3[0[ Plant material
Cell cultures of T[ chinensis were as given in "Menhard
et al[\ 0887#[ The cells were cultivated in 0 l Erlenmeyer

669
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Fig[ 5[ CI!MS spectrum of taxuyunnanine C "4# as reference "a# and the product "b# synthesized from 6 and ð0\1!02CŁacetyl CoA by the 019!fold
puri_ed acetyltransferase in the standard enzyme incubation system[
~asks containing 399 ml cell suspension over a period of
03 days at 13>C on a gyratory shaker "099 rpm# under
constant illumination "549 lux#[ An inoculum of 069 ml
suspension was added to 129 ml of fresh medium and
three days later elicited with 29 mM methyl jasmonate
"Serva#[ After seven days of cultivation the cells were
harvested by suction _ltration yielding 69 g fresh mass[
The tissue was shock frozen with liquid nitrogen and
could be stored at −19>C for up to one month[
3[1[ Chemicals
ð1!03CŁNaAc and ð0!03CŁacetyl CoA were purchased
from Amersham[ ATP\ acetyl CoA and acetyl CoA syn!
thetase were obtained from BoehringerÐMannheim[ ð0\1!
02C1ŁNaAc was purchased from Isotec[ Tert!butyl!
methylether was obtained from Fluka[ Materials for
chromatography and pre_lled columns were purchased
from Pharmacia "HiTrap Blue\ Phenylsepharose fast

B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
660
Fig[ 6[ CI!MS spectrum of baccatine III "3# as reference "a# and the product "b# synthesized by the 019!fold puri_ed acetyltransferase from 09!
desacetylbaccatine III "2# and ð0\1!02CŁacetyl CoA[#
~ow\ Mono P HR 4:19\ Sephadex G14\ DEAE!Sephacel\
PD09#\ Biorad "HighQ\ CHT II\ Bioselect SEC 149!4#\
A.nity Chromatography Ltd[ "Mimetic Green 0A5 XL#
and Serva "AcA33 Ultrogel#[ Calcium phosphate gel was
prepared as described by "Colowick\ 0844#[
Labelled acetyl CoA was prepared by incubating the
following substances for 2 h] 1 ml 4 mM Tris\ pH 6[4\ 19
mmol MgCl1 "3[95 mg#\ 04 mmol ATP "8[97 mg#\ 8[2
mmol CoAÐSH "6[53 mg#\ 2 U acetyl CoA synthetase\
8[2 mmol NaAc "9[67 mg# and 9[4 mCi ð1!03CŁNaAc "49
mCi mmol−0#[ The puri_cation and isolation of the result!
ing acetyl CoA was done by paper chromatography using
Whatman paper No[ 2 and the solvent system BuOHÐ
AcOHÐH1O "4]1]2#[ Acetyl CoA showed a Rf value of
9[15[ ð0\1!02C1ŁAcetyl CoA was prepared from ð0\1!
02C1ŁNaAc as given above[

661
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Table 1
Rate of acetylation of di}erent taxanes in the presence of acetyl CoA]09!hydroxytaxane O!
acetyltransferase "099)ꢁ5[2 nmol = s−0 = mg protein−0
#
Substrate
Relative activity ")#
09!Desacetyltaxuyunnanine C "6#
09\03!Desacetyltaxuyunnanine C "7#
4\09\03!Desacetyltaxuyunnanine C "8#
1\09\03!Desacetyltaxuyunnanine C "09#
09!Desacetylbaccatine III C "2#
099
091
77
70
79
1\4\09\03!Desacetyltaxuyunnanine C "00#
65
For the synthesis of desacetylated derivatives of 4\ in
each of _ve Eppendorfþ caps 4 "04 mg# was dissolved in
methanol "499 ml#[ NaOH "0 ml\ 9[0 N or 0 N# was added[
These mixts were incubated at 29>C for various times and
then extracted into tert!butylmethylether[ The organic
layers were combined\ dried over Na1SO3 and evapd to
dryness[ The residue was sepd using semipreparative
HPLC "Vertex RP!07\ 4 mm\ 05×149 mm\ Knauer#[
The solvent system A consisted of MeCNÐH1O "1]7# and
solvent system B of MeCN[ Gradient] 9 min 19) B^ 29
min 69) B\ 34 min 69) B^ ~ow rate] 4 ml min−0^ detec!
tion] 116 nm#[ By this method\ eight di}erent desa!
cetylated derivatives of 4 could be isolated[ Their
structures were determined by NMR spectroscopy[
The following substances were kind gifts of Indena
Comp[\ Milan] 09!DAB III "2# baccatine III "3#\ 08!OH!
09!DAB III\ 09!epi!DAB[ 1\4!Desacetyl!09\03!desa!
cetoxy!taxuyunnanine C was prepared from 4 by Dr[ U[
Maier of this laboratory[
boric acid:NaOH\ pH 7[4\ 19 mM MeSH\ 19) glycerol
"bu}er A# until the mixture was thawed[ This homo!
genate was _ltered through cheese cloth and centrifuged
at 09\999g for 09 min[ The supernatant was mixed with
Ca2"PO3#1 "0[8 g dry wt according to Colowick "0844#\
suspended in 49 ml bu}er A#[ After 09 min\ this sus!
pension was centrifuged at 2999g for 4 min and the super!
natant used for further puri_cation[
3[4[ Ammonium sulfate precipitation
The supernatant was subjected to "NH3#1SO3 pre!
cipitation "9Ð69)#[ The ppt was dissolved in 29 ml 49
mM TrisÐHCl\ pH 7[4\ 19 mM MeSH\ 19) glycerol
"bu}er B# and subsequently desalted using a Sephadex
G14 column "1[6×6 cm#[ Subsequently\ colored impurit!
ies were partly removed by a DEAE!Sephacel column
"1[4×4 cm#[ Both columns had been previously equi!
librated with bu}er B[ The protein!containing fractions
were collected and concd in a stirring cell "Amicon# to
approximately 4 ml by ultra_ltration "PM09 membrane#[
3[2[ Enzyme assay
The activity of the acetyl CoA]09!hydroxytaxane O!
acetyltransferase was measured using an incubation mix!
ture consisting of a su.cient amount of enzyme\ 49 ml
9[7 M TrisÐHCl\ pH 7[4\ acetyl CoA "4 nmol\ containing
9[91 mCi ð0!03CŁacetyl CoA# and 6 "04 nmol# dissolved in
4 ml DMSO in a total volume of 074 ml[ Assays with
boiled enzyme or DMSO instead of 6 served as controls[
After incubation for 29 min at 24>C\ the reaction was
stopped by the addition of 599 ml tert!butylmethylether
and 19 ml 09) H1SO3 and the mixture was shaken for 09
min[ After centrifugation\ the amount of 4 formed was
measured in a 499!ml aliquot of the organic layer[ This
aliquot was dried by a stream of air and the radioactivity
measured using a liquid scintillation counter or analyzed
by radio TLC "solvent system CHCl2ÐMeCN\ 6]2#[
3[5[ AcA33 gel _ltration
The concentrate was applied to an AcA33 column
"1[7×099 cm# previously equilibrated with bu}er B[ This
bu}er was also used for fractionation at 19 ml h−0
Fractions of 5[4 ml were collected and assayed for protein
and enzyme activity[
3[6[ HighQ ion exchange chromatography
The fractions of the gel _ltration step containing the
major activity were pooled "59 ml# and applied to a
HighQ column "4 ml# that had been equilibrated with
bu}er B[ The column was operated at 1 ml min−0 and
washed with 69 ml bu}er B prior to protein elution[ The
protein was eluted with a 9[96Ð0 M KCl gradient in bu}er
B "9[96 M for 09 min\ 9[03 M for 09 min\ 0 M for 04
min#[ Fractions of 0 ml were collected and assayed for
protein and enzyme activity[
3[3[ Preparation of crude enzyme extracts
All steps were performed at 3>C[ Frozen cells of T[
chinensis suspension cultures "199 g fr[ wt# were homo!
genized in the presence of 19 g PVPP\ 249 ml 099 mM

B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
662
3[7[ CHT II "hydroxylapatite#
bow markers\ Amersham# were used in order to estimate
the molecular mass of acetyl CoA]09!hydroxytaxane O!
acetyltransferase under denaturating conditions[
Active fractions "3 ml# of the HighQ column were
pooled and applied to a CHT II "4 ml# column previously
equilibrated with 09 mM NaPi bu}er pH 5[7\ 19 mM
MeSH\ 19) glycerol and operated at 9[4 ml min−0 The
column was washed with 19 ml of the same bu}er and
eluted with 04 ml of 059 mM NaPi bu}er\ pH 5[7\ 19 mM
MeSH\ 19) glycerol[ Fractions of 9[4 ml were collected
and assayed for protein and enzyme activity[
3[02[ Native molecular mass determination by gel _ltra!
tion chromatography
The molecular mass of the native enzyme was deter!
mined by gel _ltration chromatography on a Bioselect
SEC 149!4 column at 9[1 ml min−0[ The following pro!
teins were used as standards] chymotrypsinogen A "14
kDa#\ bovine serum albumin "56 kDa#\ aldolase rabbit
"059 kDa#\ catalase bovine "139 kDa#\ ferritine horse "349
kDa#[
3[8[ HiTrap Blue af_nity chromatography
The pooled active fractions "0[4 ml# were applied to a
HiTrap Blue column "0 ml# that had been equilibrated
with bu}er B[ The loading was done at 9[4 ml min−0[ The
column was washed with 04 ml bu}er B before eluting
the protein using a linear 9Ð0 M KCl gradient at 9[4 ml
min−0 "total volume 04 ml#[ Fractions of 9[4 ml were
collected and assayed for protein and enzyme activity[
3[03[ Isoelectric point
The isoelectric point of acetyl CoA]09!hydroxytaxane
O!acetyltransferase
was
determined
by
chro!
matofocussing on a Mono P HR 4:19 column "9[4
cm×19 cm# performed at 9[6 ml min−0[ Equilibration
was done with 14 mM imidazole:HCl\ pH 6[3\ containing
19 mM MeSH[ The adsorbed protein was eluted with
polybu}er 63!H1O "0]7\ Pharmacia# adjusted to pH 3
and containing 19 mM MeSH[ Fractions of 0 ml were
collected and assayed for enzyme activity[
3[09[ Phenylsepharose hydrophobic interaction chroma!
tography
Active fractions "0[4 ml# of the HiTrap Blue step were
pooled[ With a solution of 0 M "NH3#1SO3 in bu}er B
this pool was adjusted to 9[4 M "NH3#1SO3 and then
applied to a Phenylsepharose column "0×0[2 cm#[ The
column was operated by gravity and had previously been
equilibrated with 9[4 M "NH3#1SO3 in bu}er B[ It was
washed with 6 ml 9[4 M "NH3#1SO3 in bu}er B and the
protein was eluted with 9[0 M "NH3#1SO3 in bu}er B "09
ml#[ The eluate was collected in fractions of 9[4 ml and
assayed for protein and enzyme activity[
3[04[ Protein determination
Protein concs were determined by the method of
Bradford "0865# with bovine serum albumin as a stan!
dard[
3[05[ MS and NMR spectroscopy
3[00[ Mimetic Green 0A5 XL af_nity chromatography
MS spectroscopy was done using a Finnigan MAT
SSQ 69 "69 eV\ isobutane#[ NMR spectroscopy was per!
formed with a Bruker DRX 499 spectrometer[ For
measurement the taxanes were dissolved in CDCl2[
The fractions of the hydrophobic interaction chroma!
tography containing the major activity were pooled "1[4
ml# and desalted using a PD09 column and bu}er B[
Subsequently\ the desalted protein was applied to a
Mimetic Green column "0×0[1 cm# which was again
operated without pump[ This column had been equi!
librated with bu}er B and was washed with the same
bu}er "2 ml#[ Elution with 2 ml 9[4 M acetyl coenzyme A
gave an eluate containing exclusively the acetyl CoA]09!
hydroxytaxane O!acetyltransferase as judged by SDS:
PAGE[
3[06[ HPLC system
Merck!Hitachi "pump L!5199^ autosampler AS!3999^
detector L!3149#[
Acknowledgements
We thank Ms[ EnikoÃ!Melinda Kiss for excellent tech!
nical assistance\ Dr[ Jon Page for linguistic help in the
preparation of this manuscript\ Dr[ W[ Eisenreich\ Tech!
nical University\ Garching\ for measuring the NMR spec!
tra of desacetyl taxuyunnanine C derivatives and Dr[
H[ Gundlach for the MS spectra[ We also thank D[E[
Bombardelli\ Milan\ for his generosity in supplying valu!
able taxane derivatives[ This work was supported by SFB
3[01[ Electrophoretic analysis of enzyme purity
SDS:PAGE of the protein bands obtained from the
Mimetic Green step was performed as described by
Laemmli "0869#[ After electrophoresis\ protein was
detected by silver nitrate staining "Blum\ Beier\ + Gross\
0876#[ Molecular mass standards for SDS:PAGE "rain!

663
B[ Menhard\ M[H[ Zenk:Phytochemistry 49 "0888# 652Ð663
Hezari\ M[\ Ketchum\ R[ E[ B[\ Gibson\ D[ M[\ + Croteau\ R[ "0886#[
Archives Biochemistry Biophysics\ 226\ 074[
Hezari\ M[\ Lewis\ N[ G[\ + Croteau\ R[ "0884#[ Archives Biochemistry
Biophysics\ 211\ 326[
258 of the Deutsche Forschungsgemeinschaft\ Bonn and
in part by the Commission of the European Community[
Koepp\ A[ E[\ Hezari\ M[\ Zajicek\ J[\ Stofer!Vogel\ B[\ + La Fever\ R[
E[ "0884#[ Journal Biological Chemistry\ 169\ 7575[
Laemmli\ U[ K[ "0869#[ Nature\ 116\ 579[
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