Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition

Article abstract of DOI:10.1016/S0968-0896(00)00040-7

The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate Δ(24(25))-sterol acceptor molecules to Δ(25(27))- 24β-methyl products stereoselectively. Incubation with pairs of substrates - [²H₃-methyl]AdoMet and cycloartenol, and AdoMet and [27-¹³C]lanosterol - followed by ¹H and ¹³C NMR analysis of the isotopically labeled products demonstrated the si-face (β-face attack) mechanism of C-methylation and the regiospecificity of Δ(25(27))-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3β-hydroxyl group, a planar nucleus and a side chain oriented into a 'right-handed' structure (20R-chirality) - characteristic of the native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors (K(i) for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2nM, four orders of magnitude less than the K(m) for cycloartenol of 28 μM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (K(i) = 80 μM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a Δ(24(25)) to Δ(25(27)) 24β-methyl-SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00040-7

Bioorganic & Medicinal Chemistry 8 (2000) 925±936
Sterol C-methyl Transferase from Prototheca wickerhamii
y
Mechanism, Sterol Speci®city and Inhibition
Anil T. Mangla and W. David Nes*
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409USA
Received 30 September 1999; accepted 29 November 1999
AbstractÐThe membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic,
25(27)
2
4(25)
yeast-like alga, was found to C-methylate appropriate Á
2
-sterol acceptor molecules to Á
selectively. Incubation with pairs of substratesÐ[ H -methyl]AdoMet and cycloartenol, and AdoMet and [27- C]lanosterolÐfol-
-24b-methyl products stereo-
13
3
1
13
lowed by H and C NMR analysis of the isotopically labeled products demonstrated the si-face (b-face attack) mechanism of C-
2
5(27)
methylation and the regiospeci®city of Á
-double bond formation from the pro-Z methyl group (C27) on lanosterol. The
enzyme has a substrate preference for a sterol with a 3b-hydroxyl group, a planar nucleus and a side chain oriented into a `right-
handed' structure (20R-chirality)Ðcharacteristic of the native substrate, cycloartenol. The apparent native molecular weight of the
SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain
heteroatoms substituted for C24 and C25 or related structural modi®cations, including steroidal alkaloids, havs been used to probe
further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modi®cations of a
positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly
potent non-competitive inhibitors (K
nitude less than the K for cycloartenol of 28 mM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion inter-
mediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (K =80 mM). The combination of
i
for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of mag-
m
i
results suggests that the interrelationships of substrate functional groups within the active center of a Á2
4(25)
to Á
25(27)
24b-methyl-
SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
mechanisms and several of the responsible enzymes cat-
alyzing Á
2
4(28)
-formation have been isolated, cloned,
puri®ed and characterized.6 The phytosterol pathway
�10
Ergosterol (24b-methyl cholesta-5,7,22E-trien-3b-ol)
and related phytosterols are produced by all forms of
microorganisms, including algae.1 These natural pro-
ducts manifest many important biological functions and
activities in the form of membrane inserts and bior-
¯ows along di€erent channels in yeast and algae to
24(28)
,2
25(27)
generate Á
chains (Fig. 1).
- or Á
-C-methylated sterol side
3
,4
egulators. Ergosterol synthesis proceeds by the initial
cyclization of squalene oxide to lanosterol in fungi or to
cycloartenol in plants.1 The sterol methyl transferase
The frequency of occurrence of human infections from
yeast and related microorganisms have been increasing
over the past decade in response to a combination of
factors. For 30 years, drug companies have recognized
that the lanosterol±ergosterol biosynthetic pathway can
be a clinical target of powerful fungicides that impair a
,5
(
SMT), a slow and perhaps rate-limiting enzyme in the
extended biosynthetic sequence leading to ergosterol,
exhibits unique types of catalytic activities and
synthetic step thereby disrupting ergosterol home-
ostasis.1
1,12
There is broad-application of these fungi-
Abbreviations: SMT, sterol methyl transferase; AdoMet, S-adenosyl-l-
methionine; TLC, thin-layer chromatography; HPLC, high-perfor-
mance liquid chromatography; GLC, gas±liquid chromatography;
HEI, high energy intermediate; FPLC, fast protein liquid chromato-
graphy; NSF, non-saponi®able lipid fraction.
cides to microorganisms that are morphologically
similar to yeast, as typi®ed by the disease Protothecosis
caused by the non-photosynthetic alga Prototheca
13�15
wickerhamii.
Unfortunately, many of the antifungal
*
0
y
Corresponding author. Tel.: +1-806-742-1673; fax: +1-806-742-
135; e-mail: u0nes@ttacs.ttu.edu
agents used to treat infection block post-lanosterol steps
common to pathogen and host or they are polyene anti-
biotics, nystatin or Amphotericin B, that can complex
This study represents partial ful®llment for completion of the doc-
toral degree in Chemistry at Texas Tech University for A.T.M.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00040-7

9
26
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
with sterol in the fungal or animal membranes.1
6�19
Because these treatments may compromise the host
2
0
there is a renewed search for alternative drugs.
As part of an ongoing e€ort to develop new treatment
strategies to inhibit sterol pathways that are synthesized
by the pathogen, and are themselves not synthesized by
2
1�25
the host,
we have investigated the biosynthesis of
ergosterol in P. wickerhamii. Accordingly, we estab-
lished a signature lipid for diagnostic purposes with
respect to the operation of a cycloartenol±ergosterol
pathway in P. wickerhamii and identi®ed an enzyme that
can be targeted for drug formulation and testing. A ser-
ies of substrate analogues and high energy intermediate
and product analogues suitably designed so as to prevent
completion of the normal catalytic cycle of C-methyla-
tion and inhibit growth were tested with a cell-free SMT
and intact cell cultures. 25-Azacycloartenol and related
ammonium-containing sterol analogues, including the
were
steroidal alkaloids solanidine and solasodine,2
2,23
found to be potential lead compounds in drug design to
inhibit growth of this pathogenic alga. The P. wick-
2
5(27)
erhamii SMT de®nes a unique family of Á
-SMTs
that are highly conserved in action mechanism between
algae and vascular plants but absent from yeast, which
2
4(28)
20,26
produce exclusively the family of Á
-SMTs
SMTs are absent from animals that synthesize cholesterol.
and
Figure 1. Hypothetical cycloartenol-lanosterol bifurcation to ergos-
terol in fungi and algae.
Results
Sterol composition of Prototheca wickerhamii strain
YB4330
wickerhamii operates a novel cycloartenol-ergosterol
pathway (Fig. 1).
In our earlier study we examined P. wickerhamii strain
,27
2
Y-6870. In this study we examined strain YB-4330.
The main di€erence between the strains is the high level
3
Feeding experiments with [2- H]acetate
3
of protothecasterol (24b-methyl cholesta-5,7,22E,
The fate of the tritium label associated with [2- H]ace-
3
25(27)-tetraen-3b-ol) and no signi®cant ergost-7-enol
tate was determined by incubating 5 mCi [2- H]acetate
synthesized by strain Y-6870 compared with the pro-
duction of high levels of ergost-7-enol and no signi®cant
protothecasterol by strain YB-4330. As found in pre-
liminary studies using GC±MS, the major sterols of
strain YB-4330 were ergosterol and ergost-7-enol in a
ratio of about 2 to 1 (ca. greater than 95% of the
total sterols). Trace amounts of the 4,4-dimethyl
sterols cycloartenol, cyclolaudenol and 24(28)-methy-
lenecycloartanol, were detected in a ratio of ca.
with P. wickerhamii cells in the presence and absence of
35 nM 25-azacycloartenol and analyzing the resulting
sterol compositions for radioactivity. Approximately
20% of the radioactivity added to the culture medium
was found in the non-saponi®able lipid fraction. From
3
the TLC plate, three bands were found to possess H-
radioactivity corresponding to 4-desmethyl, 4-mono
methyl and 4,4-dimethyl sterols. In the control, ca. 80%
of the total radioactivity was associated with 4-des-
methyl sterols whereas in the 25-azacycloartenol treated
cells ca. 65% of the total radioactivity accumulated on
the TLC plate matching the 4,4-dimethyl zone. An ali-
quot of the non-saponi®able lipid fraction from control
incubation was injected into the HPLC27 and radio-
activity were monitored for each 30-s fraction. Mass
corresponding to two HPLC peaks of radioactivity
eluting between 15 and 18 min and representing 90% of
the applied radioactivity was coincidental with HPLC
peak retention times of ergosterol and ergost-7-enol.
Alternatively, HPLC-radiocounting analysis of the
non-saponifable lipid fraction from the 25-azacy-
cloartenol-treated cells indicated that cycloartenol was
the major sterol labeled (data not shown), consistent
with the TLC data.
1:1:0.1. From a preparative scale incubation, ®ve
sterols with chromatographic mobilities corresponding
to ergosterol, ergost-7-enol, cycloartenol, cyclolaudenol
and 24(28)-methylene cycloartanol were isolated and
puri®ed to homogeneity. Structural determination was
1
based on a comparison of the mass and H NMR spec-
tra of each compound with that of authentic specimens
available to us. The con®guration of the C24-methyl
group in ergosterol, ergost-7-enol and cyclolaudenol
1
13
was con®rmed by H and C NMR to be b-oriented
and in each case there was no evidence for contamina-
28�30
1
tion from the a-epimer.
minor 4,4-dimethyl sterol identi®ed as 24(28)-methylene
By H NMR analysis the
cycloartanol was found not to be contaminated by the
2
3(24)
25,26
ole®n with a Á
-bond.
The results show that P.

A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
927
Properties of the SMT
structure followed by isomerization of the exo methyl-
ene group to form a 24b-methyl Á
25(27)
-side chain.
The apparent molecular weight of the soluble SMT,
estimated by chromatography on a calibrated (relative to
BioRad Gel Filtration standards) Super 6 FPLC column
From a preparative scale incubation of AdoMet paired
13
13
with [27- C]lanosterol was isolated [27- C]24b-methyl
lanosta-8,25(27)-dien-3b-ol. The mass spectrum of
eluted with 50 mM Tris HCl, 2 mM MgCl , 2 mM b-
2
13
mercaptoethanol, 0.2% emulphogen and 15% glycerol
(
[27- C]24b-methyl lanosta-8,25(27)-dien-3b-ol possessed
a 1-mass unit increase (M+ 441) in molecular weight
compared with the molecular weight of the non-iso-
topically 13C-labeled ole®n (M+ 440). For [27- C]24b-
methyl lanosta-8,25(27)-dien-3b-ol, ions in the high
pH 8.0) at 0.5 mL/min, was 154,000, suggesting a mul-
timer. The pH optimum was about 7.5 (with one-half
maximal activity at pHs 7.0 and 9.5). The apparent
native molecular weight of the P. wickerhamii enzyme
predicts a monomer of 38.5 kDa, which is in the range of
13
+
mass region were found at M 441, 426, 408, 400, 383,
monomeric units (38 to 43 kDa) of related SMTs.6
�10
300, 359, 353, 345, 317, 272 and 259 amu; H NMR
spectrum: d 3.242 (H-3, 32;dd, J=4.7/11.5 Hz), 0.0.683
1
(
H-18, s), 0.810 (H-31, s), 0.874 (H-32, s), 0.910 (H-21, d
General features of sterol methyl transferase reaction
J=6.0 Hz), 0.980 (H-19, s), 1.000 (H-30, s), 1.044 (H-28,
d, J=9.5 Hz), 1.620 (H-26, d, J=5.9 Hz), 4.662 (H-27,
d, J=154 Hz). The regiospeci®c proton elimination at
the cis terminal methyl (C27) group was established by
the character and position of the chemical shifts in the
In a typical preparative experiment with P. wickerhamii
3
SMT incubated with saturating levels of [ H -methyl]A-
3
6
doMet and cycloartenol, a total of 2.9Â10 dpm was
recovered from the non-sapon®able lipid fraction and a
6
13
total of 2.4Â10 dpm was recovered from the 4,4-dime-
C NMR spectrum involving an enhanced signal at d
1
thy TLC band. Two C-methylated products were detec-
ted by HPLC of the 4,4-dimethyl sterol fraction
corresponding in chromatographic mobility to cyclo-
109 ppm for C27 and in the H NMR spectrum at d
5
laudenol (6.9Â10 dpm) and 24(28)-methylenecy-
3
cloartanol (3.4Â10 dpm), respectively. For routine
assays involving the standard preparation, conversion
of cycloartenol to 24(28)-methylenecycloartanol was
considered negligible. We have assumed, therefore, that
the C-methylation pathway involving 24(28)-methylene-
cycloartanol can be ignored in the analysis of the kinet-
ics of cyclolaudenol formation.
From a preparative incubation with cycloartenol and
AdoMet was isolated cyclolaudenol. The structure of the
SMT enzyme generated C24b-methyl product was con-
1
®
rmed by HNMR (d ppm, 0.956 (s, H18), 0.555 (d, Ha
endo), 0.330 (d, H exo), 0.859 (d, H21), 1.640 (br. s., H26),
b
4.655 (br. s., H27), 0.995 (d, H28), 0.967 (s, H30-a methyl
group), 0.810 (s, H31-b-methyl group), and 0.885 (s, H32)).
The H NMR signals of the P. wickerhamii sample were
in good agreement with that of an authentic specimen
1
2
9
available to us. These ®ndings indicate that the facial
stereochemistry of C-methylation proceeds stereo-
speci®cally from the b-face of the 24,25-double bond.
2
From a preparative scale incubation with [ H -methyl]
3
AdoMet paired with cycloartenol was isolated
2
[28- H ]cyclolaudenol. The structure was con®rmed by
the molecular weight of the product showing three deu-
3
+
terium atoms incorporated into the side chain (M 443
and other diagnostic ions at the high mass end of 428,
1
2
25, 410, 382, 355, 341, 327, 315, 303, 297 amu) amd H
NMR spectrum indicating the incorporation of the
deuterated methyl from AdoMet at C28 in the side
chain (d ppm, 0.956 (s, H18), 0.555 (d, H endo), 0.330
a
(
(
d, H exo), 0.859 (d, H21), 1.640 (br. s., H26), 4.655
br. s., H27), 0.995 (doublet for H28 missing), 0.967 (s,
b
H30-a methyl group), 0.810 (s, H31-b-methyl group),
and 0.885 (s, H32)). The deuterium introduced at C28
was established by the disappearance of the methyl sig-
nal corresponding to C28 in the spectrum. These ®nd-
ings rule out the initial formation of a 24(28)-methylene
Figure 2. Partial 1H and 13C NMR (inset to right) spectra of 24b-
methyl [27-13C]lanosta-8,25(27)-dienol isolated from an incubation of
13
the P. wickerhamii SMT and [27- C]lanosterol. The numbering sys-
tem used is shown on the sterol structure at the top of the ®gure. See
text for full details of the spectral analysis.

9
28
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
4
.672 ppm (split signal) for the H27 ole®nic proton and
3-OMe cycloartenol, only cycloartenol was catalyzed by
the SMT under the assay conditions used here (Fig. 4,
Table 1). These results indicate that the C3-hydroxyl
group and its b-orientation are necessary features for
recognition. Converting the hydroxyl group to the 3-
keto or C3-methyl ether derivative impaired recognition
thereby suggesting that added bulk at C3 a€ects the
hydrogen-bonding abilities of the C3-hydroxyl group.
Consistent with these observations was the ®nding that
removal of an a-methyl group neighboring C3 at C4, as
with 31-norcycloartenol, or complete removal of the C4-
methyl groups, as with 30,31-dinorcycloartenol, pro-
duced substrates of less competency than the parent
cycloartenol. Thus steric bulk from an angular C4
methyl group can also a€ect the recognition of the sterol.
at d 1.640 ppm (split signal) for the H26 methyl group
1
3
(
Fig. 2). The structure of the C-labeled 24b-methyl
lanosterol derivative is new. The stereochemistry at C24
of the new sterol is based on a comparison of the che-
mical shift for the H21 and H28 doublets associated
with 24a- and 24 b-methyl sterols containing a Á
sterols containing a Á
25(27)
-
2
5(27)
28
-sterol side chain.
Classi®cation of e€ects of structural modi®cations on
SMT activity
Cycloartenol was assumed to be the preferred substrate
based on the natural occurrence of cycloartenol in the cells
of P. wickerhamii. A soluble SMT was used to assess
structure±activity relationships. Dependence of C-methyl
product formation on the sterol and AdoMet concentra-
tions was found to obey Michaelis±Menten kinetics (data
not shown) with apparent Km and Vmax values for
cycloartenol and AdoMet of 28mM and 36pmol/min/mg
and 10mM and 13pmol/min/mg protein, respectively. As
shown in Table 1, no sterol was superior to cycloartenol in
The second domain examined was the nucleus (Table 1).
To assess the importance of the 9b,19-cyclopropyl feature
terms of substrate binding (K ) or in catalytic competency
m
(% transformation of substrate measured relative to the
apparent Vmax/K ratio of cycloartenol).
m
By examining the magnitude of the Km values and the
ratio of Vmax to Km of a series of sterols structurally
related to cycloartenol, it has been possible to determine
the relative structural requirements necessary for recog-
nition and to measure the substrate acceptability for
catalysis. Four domains of the cycloartenol structure
were evaluated for their relevance to sterol binding (Fig.
3). The ®rst structural domain examined involves the C3-
hydroxyl group. Of the ®ve sterols, cycloartenol, 3-deso-
xycycloartenol, 3-epicycloartenol, 3-ketocycloartenol or
Figure 3. Domains on the cycloartenol substrate investigated for rele-
vance to SMT recognition.
Table 1. Catalytic competence of sterols and substrate analogues to the SMTa
Substrate
K
m
(mM)
V
max (pmol/min)
V
max/K
m
% Transformations of substrate
compared to cycloartenol
Cycloartenol (1)
28
61
97
80
176
100
63
36
29
2
62
35
9
7
2
7
10
1.28
0.48
0.02
0.78
0.20
0.09
0.11
0.01
0.18
0.08
0.09
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
100
37
2
60
16
7
9
1
14
6
31-Norcycloartenol (2)
30,31-Dinorcycloartenol (3)
Parkeol (4)
Lanosterol (5)
Cholesta-5,20(22)E,24-trienol (6)
Desmosterol (7)
Cholesta-5,17(20)E,24-trienol (8)
Zymosterol (9)
276
38
14a-Methylzymosterol (10)
Cholesta-5,7,22E,24-tetraenol (11)
125
112
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10
7
3
3
3
3
-Epicycloartenol (12)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
-Desoxycycloartenol (13)
-OMe Cycloartenol (14)
-Keto-Cycloartenol (15)
Cholest-8-enol (16)
0a-Cucurbitacin (17)
Tirucallol (18)
0-Epidesmosterol (19)
1
2
Cholesta-5,17(20)Z,24-trienol (20)
Cholesta-5,25(27)-dienol (21)
Cycloartanol (22)
2
4-Methyldesmosterol (23)
aSee text for details of enzymatic assay. Structures of the sterols are given in Figure 4. ND means not determined due to low or nonexistent of
transmethylation.

A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
929
Figure 4. Substates tested and their corresponding catalytic competence (Vmax//K
norcycloartenol; 3, 30,31-Dinorcycloartenol (24-dehydropollinstanol); 4, parkeol; 5, lanosterol; 6, 20(22)E-dehydrodesmosterol; 7, desmosterol; 8,
7(20)E-dehydrodesmosterol; 9, zymosterol; 10, 14a-methylzymosterol; 11, cholesta-5,7,22E,24-tetraenol; 12, 3-epicycloartenol; 13, 3-desoxy
m
) toward the P. wickerhamii SMT enzyme. 1, cycloartenol; 2, 31-
1
cycloartenol; 14, 3-OMe cycloartenol. 15, 3-ketocycloartenol, 16, cholest-8-enol (24-dihydrozymosterol) 17, 10a-cocurbitacin; 18, tirucallol; 19, 20-
epidesmosterol; 21, 17(20)Z-dehydrodesmosterol; 21, cholesta-5, 25(27)-dienol; 22, cycloartanol; 23, 24-methyl desmosterol.
5
8
in the cycloartenol nucleus, we chose to compare cycloar-
tenol with four related structural isomers of cycloartenol,
parkeol, lanosterol, tirucallol and 10a-cucurbitacin. The
activities of desmosterol (Á ) versus zymosterol (Á ) are
consistent with the conclusion that the position of the
nuclear bond is important for optimal substrate recogni-
tion. Thus the relative importance of nuclear features in
binding measured as Km values are: 9b,19-
9
(11)
ability of parkeol with a Á
-bond to bind to the SMT
indicated that the 9b,19-cyclopropane ring was not essen-
tial to competency. However, placement of the double
9
8
5
cyclo<Á <Á <Á ; 4,4-dimethyl<4-monomethyl<4,
8
bond in the Á -position generated a sterol that is less
4-desmethyl.
competent than parkeol. Inversion of the con®guration of
lanosterol at C13, C14 and C17, producing tirucallol,31 or
changing the nuclear conformation from ¯at (in cycloar-
The third domain studied was the stereochemistry at
C20 and the side chain conformation. To test the sig-
ni®cance of the C20 chirality on sterol binding cap-
abilities of ®ve structurally similar sterols were compared
desmosterol (20R-cholesta-5,24-dienol), 20-epidesmos-
terol (20S-cholesta-5,24-dienol), 17(20)E-dehydrodes-
mosterol (cholesta-5,17(20)E,24-trienol), 17 (20)Z-dehy-
drodesmosterol (cholesta-5,17(20)Z,24-trienol) and 20
(22)E-dehydrodesmosterol (cholesta-5,20(22)E,24-tri-
enol). We chose to examine the desmosterol series of
compounds since the relevant cycloartenol series of
compounds was not available. Desmosterol was recog-
nize by the SMT with an apparent Km 63 mM in the
same order of magnitude of cycloartenol.
2
3
32
tenol) to bent (10a-cucurbitacin, led to a less com-
petent structure than lanosterol. These results suggests
that the position of the double bond and the three-
dimensional shape of the nucleus a€ects recognition. It
is possible that some non-substrates that were tested
bind weakly to SMT but are not methylated, or methy-
3
lated slowly. In support of this view, [3- H]lanosterol and
3
[3- H]cholest-8-enol assayed with the pure recombinant
yeast SMT failed to show productive binding (transfor-
2
9
mation), but nevertheless, was found to bind weakly to
the enzyme; K of lanosterol and cholest-8-enol was about
d
two orders of magnitude less than the Km of zymosterol
(
unpublished). The angular 14a-methyl group was found
to minimally impair recognition based on a comparison of
the activities of zymosterol and 14a-methyl zymosterol
toward the P. wickerhamii SMT. A comparison of the
Under the assay conditions used for these experiments,
desmosterol, cholesta-5,20(22)E,24-trienol, and cho-
lesta-5,17(20)E,24-trienol (K =63 mM, 100 and 276 mM,
m

9
30
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
respectively) were catalyzed by the SMT whereas neither
0-epidesmosterol or cholesta-5,17(20)Z,24-trienol were
e€ectively transformed by the SMT. The results indicate
that the natural con®guration of 20R is critical but not
obligatory to sterol binding (as con®rmed from the study
2). The ability of 20(22)E-dehydrodes-mosterol and
17(20)E-dehydrodesmosterol to bind to the SMT proves
that the C20 taetrahedral character, native to cycloarte-
nol, is not essential for binding. Nonetheless, the side
chain must orient into the right-handed conformation
for optimal catalytic competency. Cholesta-5,7,22E,24-
tetraenol was bound to the SMT similar to 20(22)E-
dehydrodesmosterol, suggesting that the side chain con-
formation assumes a staggered conformation from C20
to C26 for transformation.
2
of the binding constants (K ) determined for the epimeric
i
pair of inhibitors 20R- and 20S-22-azacholesterol (Table
Table 2. Blockage of SMT activity by substrate analogues and
rationally designed intermediate inhibitorsa
The fourth domain examined was the Á24(25)-bond. The
importance of a Á24(25)-bond in the sterol side chain was
demonstrated by comparing the activities of cycloar-
tenol with cycloartenol and zymosterol and cholest-8-
enol. No transformation was evident with either
Inhibitor
K
i
K
i
/K
m
Kinetic
pattern
1.1Â10�
4
4
4
4
4
4
3
4
NC
NC
NC
NC
NC
NC
NC
NC
C
C
C
C
C
2
2
2
2
2
2
2
2
2
5-Azacycloartenol (24)
5-Azalanosterol (25)
5-Azacholesterol (26)
4-Azazymosterol (27)
3-Azacholesterol (28)
0S,22-Azacholesterol (29)
4(R,S),28-Epiminocholesterol (30)
4(R,S),25-Epiminolanosterol (31)
0R,22-Azacholesterol (32)
3 nM
7 nM
�
�
�
�
�
�
�
0
2.5Â10
10 nM
15 nM
20 nM
25 nM
85 nM
13 nM
50 mM
2 mM
75 mM
23 mM
85 mM
80 mM
ND
3.6Â10
5.6Â10
cycloartenol or cholest-8-enol as substrate. Regiospe-
24(25)
7.1Â10
ci®cty for the Á
-bond was demonstrated by
8.9Â10
comparing the activities of desmosterol and cholesta-
5,25(27)-dienol. No transformation was evident with
cholesta-5,25(27)-dienol as substrate. Bulk at C24 inter-
feres with C-methylation activity as shown by the e€ec-
tive transformation of desmosterol (24H-atom) and the
lack of transformation of 24-methyl desmosterol (24-
methyl group). 24(28)-Methylene cycloartenol (Table 2,
Fig. 5) was not transformed under the assay conditions
tested, suggesting that the algal SMT most likely will
not perform the consecutive C-methylations of
cycloartenol to 24-ethylidene cycloartanol during active
cell proliferation.
3.0Â10
4.6Â10
1.78Â10
0
Solanidine (33)
Solasodine (34)
Cyclolaudenol (35)
0.7Â10
0
2.7Â10
0
0.8Â10
0
2
4(28)-Methylenecycloartanol (36)
3.6Â10
0
Ergosterol (37)
Cholesterol (38)
Sitosterol (39)
2.9Â10
C
ND
ND
ND
ND
ND
aSee text for details of enzymatic assay. Structures of the sterols are
given in Figure 5. K
m
is that of cycloartenol; C-competitive; N.C.,
non-competitive; N.D., no activity determined.
Figure 5. Inhibitors tested (range from 1 nM to 100 mM) with the SMT from P. wickerhamii. Cycloartenol and AdoMet were added at a ®xed con-
centration of 50 mM. Compounds shown are: 24, 25-azacycloartenol; 25, 25-azalanosterol; 26, 25-azacholesterol; 27, 24-azazymosterol; 28, 23-aza-
zymosterol; 29, 20S,22-azacholesterol; 30, 24(R,S),28-epiminofucosterol; 31, 24(R,S),25-epiminolanosterol; 32, 20R,22-azacholesterol; 33,
solanidine; 34, solasodine; 35, cyclolaudenol; 36, 24(28)-methylenecyclartanol; 37, ergosterol; 38, cholesterol; 39, sitosterol.

A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
931
High energy intermediate analogues
molecule. Another possibility is that carbenium ion
character is developed in a concerted reaction such that
C-24-alkylation and C-27 proton elimination are
simultaneous thereby eliminating the possibility of the
formation of a C25 cation during C-methylation.
To help de®ne the structural requirements of the SMT
active site, high energy intermediate analogues with a
nitrogen substitutent were examined for their ability to
inhibit the SMT-catalyzed reaction in the direction of
cyclolaudenol formation. A list of potential inhibitors
The steroidal alkaloids solasodine and solanidine with
the ammonium group caged in a ring system were tested
and found to inhibit SMT activity in the micromolar
range. The 22-azacholesterol sample with the unnatural
con®guration at C20R (20a-hydrogen atom) was also
found to bind to the SMT but much less eciently than
22S-azacholesterol with the natural C20-con®guration
(20b-hydrogen atom). The molecular parameters of the
inhibition indicate that the sterol side chain may bind to
the SMT with di€erent structural parameters (open
chain versus caged) or in di€erent orientations (C20R or
C20S-alternate con®gurations that a€ect the side chain
conformation). The nature of these interactions suggest
that an anionic center exists in the active site in the
vicinity of C24/C25 of the native substrate correspond-
ing to the 24-methyl 24(25)-carbenium ion intermediate
formed during C-methylation of the 24,25-double bond.
In order to interact with the anionic center (deproto-
nating base) optimally the side chain must orient to the
``right-handed'' conformation.31
(
tested to a concentration of 100 mM) and a summary of
inhibition characteristics are reported in Table 2 (cf.
accompanying structures in Figure 5). For mechanistic
reasons, we examined a series of ammonium derivatives
in which an azridine group or aza group was introduced
in the lateral chain between carbon atoms C22 and C25.
Most of the sterol analogues were potent inhibitors of
the SMT activity exhibiting non-competitive type
kinetic patterns with low K values in the nM range.
i
The e€ectiveness of the sterol analogues on inhibition of
the SMT activity was found to be related to the nuclear
structure of the inhibitor and on the structure of the side
chain. The sterol analogues were found to group into
three groups related to their kinetic patterns and degree
of inhibitory power. Group I (Table 2: 24±31) contains
compounds that exhibited non-competitive type kinetics
with respect to cycloartenol whereas Groups II (32±35)
and III (36±39) contained compounds that exhibited
competitive-type kinetics. Group I inhibited in the
nanomolar range whereas Groups II and III inhibited in
Product analogues and related inhibitors
the micromolar range, similar to the K of cycloartenol.
m
None of the inhibitors were found to exhibit time-
dependent inactivation type kinetics, suggesting they
were all reversible inhibitors. The expectation from a
non-competitive-type kinetic pattern is that binding of
the inhibitor occurs at a site on the enzyme other than
the site for the normal substrate. The high potency is
considered to result from tight-binding and from the
ammonium-containing side chain to interact at a subsite
To determine the inhibition from product analogues
(Fig. 5), 24(28)-methylene cycloartanol was compared
to cyclolaudenol, and ergosterol was tested relative to
sitosterol and cholesterol. 24(28)-Methylenecycloartanol
was an e€ective competitive inhibitor of SMT enzyme
activity with a K of 85 mM, whereas the K of the com-
petitive inhibitor cyclolaudenol was 23 mM. The greater
ecacy of binding of cyclolaudenol compared to 24(28)-
methylene cycloartanol is consistent with the C-methyl
mechanism to generate a 24b-methyl sterol. Ergosterol
was also a competitive inhibitor of SMT activity with a
i
i
in the active center that di€ers from the location nor-
mally occupied by the Á2
4(25)
-bond at initial binding.
The relative binding eciency of 25-azacycloartenol, 25-
azalanosterol and 25-azacholesterol follows the order of
K of 80 mM whereas neither cholesterol nor sitosterol
i
were found to inhibit SMT activity, even at the highest
concentration of inhibitor tested of 200 mM. The speci-
®city for ergosterol to down-regulate SMT activity
indicates the binding pocket of the SMT distinguishes
the size of the group at C24.
24
acceptability of the corresponding Á -sterols as substrates
for P. wickerhamii. This trend suggests that 25-azasterols
behave as sterol mimics of the SMT enzyme. Regardless of
whether the nitrogen atom was introduced at C25, or at
C24, C23 or C22, the inhibitory power of the azasterol
was about the same. 24(R,S),28-Epiminofucosterol (Ki
Inhibition of growth and sterol analysis
8
2
5 nM) was much less e€ective at binding than
4(R,S),25-epiminolanosterol (K =13 nM), con®rming
25-Azacycloartenol, solasodine and ergosterol were tes-
ted as inhibitors of growth. Only the nitrogen-contain-
ing compounds inhibited growth, presumably because
only they were actively absorbed by the cells. Inhibition
of cell growth was found to lead to cell death as deter-
mined by cell fragmentation microsopically of 25-aza-
cycloartenol treated cells. The IC50 value of cells treated
with 25-azacycloartenol was 35 nM and the IC50 value
for solasodine was 2 mM. At the IC50 concentration of
inhibitor, the sterol compositions resulting from the two
treatments were similar with cycloartenol predominat-
ing the sterol mixture. Ergosterol was the major sterol
observed in untreated P. wickerhamii, total cellular
sterol=19 fg/cell, whereas in 25-azacycloartanol- and
i
the observation with 24-methyldesmosterol, that
increased bulk at C24 inhibits C-methylation activity.
The NSF fraction from select azridine- or aza-inhibitor
treated cycloartenol incubations was examined by
HPLC.
There was no evidence of formation of 25-hydroxy
sterols by HPLC from the incubation with 25-aza-
cycloartenol, suggesting that a discrete C25 cation had
been formed during the reaction cycle involving
cycloartenol as substrate either the 25-hydroxy sterol
was not released (covalent attachment to the enzyme)
or there was not sucient time to react with a water

9
32
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
solasodine- treated cells, the total cellular sterol at IC50
was signi®cantly increased to 104 fg/cell and 372 fg/cell,
respectively. The IC50 value for 25-azacycloartanol cor-
Á24(28)- or Á25(25)-ole®ns whereas in the noncovalent
mechanism a single side chain orientation is employed
to generate the product distribution (Fig. 6). Additional
features of the steric-electric plug model of sterol C-
methylation involve a reaction mechanism whereby the
related well with the K value for inhibition of SMT
i
enzyme activity. The speci®c accumulation of cycloar-
tenol in the 25-azacycloartenol and solasodine-treated
cells con®rms the mode of action of these compounds is
to target the inhibition of the SMT enzyme activity and
that cycloartenol cannot replace ergosterol as a mem-
brane insert, similar to our earlier observations with
24
facial disposition of the Á -bond relative to AdoMet
and its counter ion promotes alkylation from the si face
of Á24 followed by migration of a hydrogen atom from
C24 to C25 across the re face and a kinetic mechanism
whereby speci®c molecular interactions develop in the
ternary complex of the active centre between key
nucleophilc groups associated with the sterol (C3
3
3,34
yeast.
24
hydroxyl group and Á -bond) and a polar amino
acid(s). Although many aspects of this model have been
veri®ed experimentally, including studies on fungal and
vascular plant SMTs involving substrate and HEI ana-
Discussion
Carbocations typical of those generated during biomi-
metic C-methyl transfer reactions of an ole®n are
logues5
0�60
and structural and kinetic analyses involving
3
5
notoriously unselective in their reactions. Nonetheless,
the ®ne-tuning of SMT enzymes under the pressure of
natural selection has resulted in ecient catalysts that is
re¯ected in the regio- and stereoselectivity to the reac-
tions they promote and in the high degree of sterol spe-
site-directed mutagenesis of the SMT sterol binding site
from S. cerevisiae,42 little is known about SMT structure
and function from primitive organisms.
Three major families of SMT proteins are speculated to
exist according to the nature of the predominant pro-
ci®city for binding.3
6�43
Two mechanisms have been
proposed for the C-methylation of a sterol acceptor
6
24(28)
duct formed from catalysis: SMTI (Á
23(24)
-ole®n),
-ole®n), all of
3
6�39,44
25(27)
molecule, a covalent (X-group)
covalent (steric-electric-plug)
and a non-
0�42
mechanism. Crucial
SMTII (Á
-ole®n) or SMTIII (Á
4
which should pass through a 24b-methyl high energy
intermediate. SMTIII can be considered a more primi-
tive SMT enzyme based on the product, 24b-methyl
to the structure and stereochemistry of the 24-methy-
lated products generated by these di€erent mechanisms
is the orientation of the sterol side chain at initial sub-
strate binding; in the covalent mechanism two side
chain conformations are employed to generate the
2
5(27)
Á
, which characterizes the sterol composition
48
24(28)
of primitive plants. SMTIII can generate a Á
4(28)
ole®n, but the Á2
-product is expected to be a minor
Figure 6. Two oppposing views of the Á24(28)- and Á25(27)-C-methylation pathway(s) operating in alga phytosterol synthesis: (A) The X-group
mechanism involves a step-wise mechanism of two di€erent sterol side chain conformations to achieve the ®nal product distribution catalyzed by the
SMT; (B) the steric±electric plug model involves a concerted mechanism of a single phytosterol side chain conformation to achieve the ®nal product
distribution catalyzed by the SMT; path b is the expected route to ole®n formation. See text for further details.

A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
933
component of the C-methylated sterol mixture. In view
of the recent demonstration that as many as three SMTs
Conclusion
(
dopsis) are formed to catalyze a Á
isoenzymes) from the same plant (tobacco and Arabi-
2
The demonstration that select HEI analogues can inhi-
bit the C-methylation step and inhibit cell proliferation
of P. wickherhamii appears to result from the initial
positioning of the C-methylation step in the cycloarte-
nol±ergosterol pathway compared to its positioning the
lanosterol±ergosterol pathway in yeast and from the
inability of cycloartenol to replace ergosterol in sterol-
controlled membrane competence thereby having a
greater disruptive in¯uence on ergosterol homeostasis in
the alga. These results contrast with a yeast or proto-
zoan system where the C-methylation step occurs late in
the lanosterol±ergosterol pathway, so that upon cellular
treatment with 25-azasteroids, zymosterol (a membrane
competent sterol) accumulates leading to reduced
4(28)
-structure with
0,45,46
1
either a C - or C -group attached to C24,
1
and the
2
observation herein that P. wickerhamii synthesizes trace
levels of 24-methylene sterols in situ, is reason that
SMTs may exist in select organisms or tissues singly or in
multiple molecular forms. One of the enzyme forms can
be bifunctional to permit the consecutive C-methylations
2
4
of the Á -bond. Assuming that SMTs exist as iso-
enzymes, they can be expected to be di€erentiated by
sterol speci®city, allosteric modulators, such as ergo-
sterol, and/or electrophoretically based on di€erent pI
values for each isoenzyme. Thus, the observation that the
P. wickerhmaii SMT prefers cycloartenol to zymosterol,
indicates a `plant-derived' SMT. Alternatively, the
observation that the activity of the P. wickerhamii SMT
is inhibited by ergosterol and not by sitosterol, suggests
that the P. wickerhamii SMT is a primitive SMT.
growth rates.5
1�58
The di€erent sensitivities of micro-
organisms toward 24-amino and 24-epimino steroids
may also be from the positioning of the SMT in the post-
lanosterol pathway. For these reasons, rational drug
design targeted for the C-methylation reaction, occur-
ring as a ®rst step in the post-lanosterol±cycloartenol
pathway may o€er a new approach to treat disease,
including Protothecosis. The search for natural pro-
ducts such as plant alkaloids solasodine, solanidine and
the marine alkaloid plakinamine and related com-
pounds57 to block SMT activity of microbes sensitive to
these compounds may also warrant a renewed interest.
The results establish conclusively that sterol competency
of the algal SMT catayzed reactions involves recognition
by both poles of the sterol molecule, similar to the action
2
8,29
from fungal and vascular plant SMTs.
Structure±
activity studies of substrate analogues tested with the P.
wickerhamii SMT indicated four molecular domains on
cycloartenol, the native substrate, were essential to bind-
ing: (i) 3b-OH group, (ii) planar nucleus, (iii) 20R-stereo-
chemistry in which the side chain is oriented to the right
The combination of results suggest that SMTIII from P.
wickerhamii may have a di€erent complement of active-
site amino acid residues involved in substrate binding
and catalysis than those of SMTs from yeast and higher
plants operating as SMTI- type enzymes. Further
investigations to de®ne the active-site structure of the P.
wickerhamii SMT should reveal the speci®c catalytic
features that are responsible for the observed di€erences
in properties and reaction pathways between the alga
SMT and related SMT enzymes.
(
and (iv) C -side chain with an intact terminal isopropyl
C22 transoid to C18 in the usual view of the molecule)
8
group and an unhindered 24,25-double bond. Of less
importance to sterol competency was substitution at C4
or the degree of unsaturation in the nucleus.
The coupled methylation-deprotonation catalyzed by
the P. wickerhamii SMT was found to be stereoselective
1
and regiospeci®c. Based on the H NMR analysis of the
enzyme-generated cyclolaudenol, the C-methylation of
2
4(25)
the Á
-bond associated with cycloartenol the methyl
group introduced at C24 was b-oriented. The retention
of three deuterium atoms at C28 from incubation with
Experimental
2
24(28)
[
H -methyl]AdoMet indicates a Á
3
-intermediate is
not involved with the reaction pathway. Thus, the C24
Algal material, substrates and reagents
methyl group must be delivered from the b-face of the
24,25-double bond, similar to the si face reaction
mechanism catalyzed by SMTs from yeast and vascular
P. wickerhamii strain YB-4330 was obtained from Dr.
C. P. Kurtzman (US Department of Agriculture/
Northern Regional Research Center, Peoria, IL). The
6
,7,49
1
13
ꢀ
plants.
Based on the H and C NMR analysis of the
cultures were grown at 29 C on a medium of 1% Bacto-
13
enzyme-generated [27- C]25(27)-24b-methyl 24,25-dihy-
drolanosterol, the regiospeci®c proton elimination occur-
red at the cis terminal (Z)-methyl group to become C27
yeast extract/2% Bacto peptone/2% dextrose. The cul-
tures (300 mL medium per 1-L Erlenmyer Flask) were
5
inoculated with 0.5Â10 cells/mL and shaken con-
8
(
CH to CH eliminations generated during C-methyla-
an exo-methylene group). The observed regiospeci®c
tinuously for 48 h to growth arrest, ca. 1.0Â10 cells/
mL, yielding per ¯ask ca. 5.7 g fresh weight of packed
cells. The cells were cultured in the dark on a New
Brunswick Scienti®c Shaker at 180 rpm. Cells were har-
vested by centrifugation (10,000Âg for 10 min) using a
J2-HS Beckman centrifuge and the resulting packed cells
3
2
tions of the 24,25-double bond to produce related 24b-
methyl or ethyl-Á -sterols investigated thus far occur
2
5
4
4,47
consistently at the cis-methyl group.
5(27)
We conclude
-bond is concerted with
2
that the formation of the Á
C-24 methylation and that the C24 con®guration of
ꢀ
stored at � 20 C. Cell counting methods were followed
cyclolaudenol should directly re¯ect the direction (i.e.,
si-face or backside attack) of methylation of the Á2
as described.21 All sterol substrates and inhibitors were
purchased commercially, isolated from natural sources
4(25)
-
2
2�25
bond of the corresponding sterol substrate, cycloarte-
nol, consistent with the steric±electric plug model.40
or synthesized as described in our earlier papers,
except for the synthesis of 20-epidesmosterol and 3-des-

9
34
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
oxycycloartenol which will be described elsewhere.
1
concentration of 0.1% (w/v)) to give a total volume in
the assay mixture of 0.6 mL and ®nal concentration of
each substrate of 50 mM, unless otherwise noted. The
protein concentration in the microsomal suspension was
maintained between 2.5 and 3.0 mg/mL. The production
of radiolabeled C-methylated sterols using the soluble
enzyme preparation was linear with respect to time for
up to 3 h at a protein concentration of 1.5 mg/mL, and
all subsequent experiments were carried out well within
these linear assay conditions. The rates of C-methyla-
tion as a function of substrate concentration with
cycloartenol gave rise to typical hyperbolic saturation
curves. The double reciprocal plots were linear and the
measured Km values for cycloartenol were about
28‹3 mM where the number of di€erent trials equaled
10. These assay conditions were found to be optimal
3
26
[
iodide salt was purchased from Sigma, [ H -methyl]A-
27- C]Lanosterol was prepared as described. AdoMet
3
3
3
doMet (10±15 Ci/mmol) and [2- H]acetate (75±150 mCi/
2
mmol) were purchased from NEN DuPont and [ H -
3
methyl]AdoMet (99.3 at % deuterium) as the p-toluene-
sulfonate salt was purchased from MSD Isotopes
(
Canada). All other biochemicals and reagents were
purchased from Sigma or Aldrich, unless otherwise
noted.
Inhibition of Growth of P. wickerhamii
Duplicate ¯asks of growth medium containing a variety
of concentrations of the chosen inhibitor dissolved in
100 mL ethanol (usually four concentrations, plus a
control and none) were inoculated with equal numbers
of cells and grown for 48 h. The approximate con-
centration of inhibitor supplied to medium for 50%
inhibition of growth was reported as the IC50 value.
Ethanol at the same concentration used to solubilize the
inhibitors had no apparent e€ect on growth.
and similar to those reported for related SMT enzymes
from fungi and vascular plants.5
0,51
The reaction was
quenched by the addition of 10% KOH in ethanol (w/
v). To increase the recovery of sterol from the quenched
reaction, we found that adding 1 mL of DMSO to the
ꢀ
quenched reaction and heating at 95 C for 10 min fol-
lowed by extraction with skelly solve F (mixed hexanes)
(
3Â1 mL) led to an increase of sterol recovered from the
Enzyme isolation, solubilization and assay
incubation mixture by about 3-fold compared to
experiments where the dimethyl sulfoxide (DMSO)
treatment was not followed (65,000 dpm per assay ver-
sus 23,000 dpm per assay). Generally, the radioactivity
associated with the DMSO-treated non-saponi®able
lipid fraction served to establish product conversion.
For some experiments, the combined hexane extracts
from DMSO-treated incubations were analyzed by TLC
to separate sterols by the degree of substitution at C4.
The desired sterol class (4-desmethyl, 4-monomethyl or
4,4-dimethyl sterol) was eluted from the TLC plate,
injected into HPLC and an aliquot of each fraction
taken for liquid scintillation counting to determine the
conversion rate or product distribution. Radioactivity
was measured in a Beckman LS #6500. Liquid scintilla-
tion counting was performed in 5 mL of Scintiverse
P. wickerhamii cells cultured to the onset of growth
arrest were used as the enzyme source for determination
of constitutive sterol methyl transferase activity. Frozen
cell pellets were added to a chilled mortar and allowed
to thaw. The mass was refrozen by adding liquid nitro-
gen. An equal amount of sea sand to the weight of the
frozen pellet was added to the mortar and the cells bro-
ken by grinding vigorously for 15 min. The resulting
paste was washed with homogenizing bu€er (bu€er A:
0.1 M Tris±HCl (pH 7.5 to 8.0), 4 mM MgCl , 10 mM
2
b-mercaptoethanol, 0.5 M sucrose and 300 mg/100 mL
bovine serum albumin) and ®ltered through four layers
of cheesecloth. The ®ltrate was clari®ed by centrifuga-
tion at 7000Âg for 20 min and further centrifuged at
1
00,000Âg for 90 min to sediment the microsomes.
3
From 53 g fr weight of cells, eight microsomal pellets
were generated. Solubilization of the SMT enzyme was
achieved by adding the combined microsomal suspen-
sion of eight pellets to a resuspension bu€er containing
(Fisher) at a H counting eciency of 56%.
For preparative incubations, the number of assays was
increased to 90±100, generating about 100 to 300 mg
product. Each assay contained the standard protein
concentration and saturating levels of AdoMet and
sterol substrates, and the incubation time was extended
to 15 h. The assays were pooled, saponi®ed and extrac-
ted in the usual manner. The resulting non-saponi®able
lipid fraction was chromatographed by TLC and HPLC
to isolate sterols to homogeneity. For the isolation of
sterols from whole cells, the combined cell pellets were
ground to a paste and saponi®ed by treatment with a
20% glycerol and the sample homogenized until the pel-
lets were evenly distributed in bu€er. The sample was
poured into a beaker on ice and emulphogen (poly-
oxoethylene 10 tridecyl ether, Sigma; 10% (v/v)) was
added to a ®nal concentration of 0.3% (volume of deter-
gent/volume of bu€er containing 20% glycerol). The
solubilized material was allowed to stand for 30min on ice
with periodic shaking. The sample was centrifuged at
1
00,000Âg for 1 h. The resulting supernatant was used to
assay the SMT. Protein determinations were as described
with bovine albumin as the standard.61
re¯uxing solution of 10% KOH, 10% H O and 80%
2
aqueous methanolic KOH (w/v/v) for 30 min. The
resulting non-saponi®able lipid fraction was chromato-
graphed on TLC and HPLC to obtain pure sterols.
The standard assay of the microsomal or solubilized
SMT activity was performed in 0.5 mL of the respective
suspension dissolved in bu€er B (50 mM Tris±HCl, pH
Inhibitor assay
7
4
.5±8.0, 2 mM MgCl and 2 mM b-mercaptoethanol) for
2
ꢀ
3
5 min at 35 C in the presence of 50 mL of [ H -methyl]
Boiled controls were included in each experiment, and
in all cases non-enzymatic product formation was neg-
ligible. Preliminary studies indicated that the proportion
3
AdoMet (0.5±1 mCi/mL), substrate or substrate analo-
gue dissolved in 50 mL bu€er A and Tween 80 (®nal

A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
935
of Á25(27)-24b-methyl product formed in the non-sapo-
ni®able lipid fraction was not in¯uenced by the degree
of inhibition with any of the analogues, thus permitting
routine determination of sterol methylation as a mea-
sure of SMT enzyme activity. The sterol to protein ratio
in the microsomes were found to be 20 mg/mg, and after
solubilzation of the SMT to be 4 mg/mg. The only sterol
detected in the soluble SMT fraction was ergosterol.
Inhibition by each analogue, and velocity comparisons
between the di€erent sterol substrates were determined
using single enzyme preparations thereby avoiding any
preparation to preparation variation. All inhibition stu-
dies were performed using variable analogue concentra-
tions in the presence of ®xed concentrations of sterol
substrate (usually cycloartenol) and AdoMet. Inhibition
7. Tong, Y.; McCourt, B. C.; Guo, D.; Mangla, A. T.; Zhou,
W-X.; Jenkins, M. D.; Zhou, W.; Lopez, M.; Nes, W. D. Tet-
rahedron Lett. 1997, 38, 6115.
8. Grebenok, R. J.; Gailbraith, D. W.; Penna, D. D. Plant
Mole. Biol. 1997, 34, 891.
9. Shi, J.; Gonzales, R. A.; Bhattacharyya, K. J. Biol. Chem.
1996, 271, 9384.
10. Bouvier-Nave, P.; Husselstein, T.; Desprez, T.; Benveniste,
P. Eur. J. Biochem. 1997, 246, 518.
1. Venkatraemsh, M.; Nes, W. D. Arch. Biochem. Biophys
1995, 324, 189.
1
12. Georgopapadakou, N. H.; Walsh, T. J. Science 1994, 264,
371.
13. Berg, D.; Plempel, M. In Sterol Biosynthesis Inhibitors:
Pharmaceutical and Agrochemical Aspects; Berg, D. and
Plempel, M. Eds.; Ellis Horwood Ltd: Chichester, 1988; pp 1±
1
1
3
1
1
45.
4. Yip, Y. Y.; Huang, C.; Clark, W. H. J. Dermatol 1976, 3,
09.
5. Tindall, J. P.; Fetter, B. F. Arch. Derm. 1971, 104, 490.
6. Kaminiski, Z. C.; Kapila, L. R.; Sharere, P.; Kliser, P.;
type and K values were determined by standard gra-
i
5
8
phical procedures, for which computer assisted linear
regression analysis a€orded correlation coecients
greater that 0.85 in all cases. For velocity determination
with the substrate analogues, the production of methy-
lated sterol was monitored as outlined above. The sam-
ple to sample variation (in dpm) was less than 10% in
all cases as judged by direct comparison of duplicate
experiments.
Kaufman, L. Clin. Infect. Dis. 1992, 15, 704.
17. Dukes, M. N. G. In Mylers Side E€ects of Drugs; Smith,
G. S., Ed; Elsevier: Amersterdam, 1980; 9th ed.; pp 727±737.
1
1
1
1
8. Hartman, P. G.; Sanglard, D. Current Pharm. Des. 1997, 3,
77.
9. Hebeka, E. K.; Solotorovsky, M. J. Bacteriol 1965, 89,
533.
Identi®cation of metabolites
20. Guo, D.; Mangla, A. T.; Zhou, W.; Lopez, M.; Jia, Z.;
Nichols, D.; Nes, W. D. Subcellular Biochem. 1997, 8, 89.
21. Nes, W. D.; Hanners, P. K.; Parish, E. J. Biochem. Bio-
phys. Res. Com 1986, 139, 410.
22. Nes, W. D.; Guo, D.; Zhou, W. Arch. Biochem. Biophys
1997, 324, 68.
23. Janssen, G. G.; Nes, W. D. J. Biol. Chem. 1992, 267,
The chromatographic and spectral properties of the
metabolite(s) from selected incubations were determined
by their rates of movement in TLC (Silica gel G plates
developed in benzene:diethyl ether (85:15), GLC (3%
ꢀ
SE-30 packed columns operated isothermally at 245 C)
2
2
1
2
2
2
3
2
5856.
4. Jia, Z.; Zhou, W.; Guo, D.; Nes, W. D. Synthetic Commun.
996, 26, 3841.
5. Guo, D.; Venkatramesh, M.; Nes, W. D. Lipids 1995, 30,
03.
6. Zhou, W.; Guo, D.; Nes, W. D. Tetrahedron Lett. 1996,
7, 1339.
7. Norton, R. A.; Nes, W. D. Lipids 1991, 26, 247.
and HPLC (two HPLC columns, Whatman C18 or
Zorbax C , were employed to separate structurally
similar compounds), by GC±MS (Hewlett-Packard 5973
1
8
1
13
mass detector) or by H and C NMR analysis (Bruker
AF300-MHz for 1H and 75 MHz for C; samples
referenced to tetramethyl silane or chloroform) as
13
described in earlier publications.2
7,62
Relative retention
times in GLC (RRt ) and HPLC (a ) are relative to the
mobility of cholesterol eluting from the column.
28. Kokke, W. C.; Pak, M. C.; Fenical, W.; Djerassi, C. Helv.
Chem. Acta 1979, 62, 1310.
c
c
2
9. Nes, W. D.; Benson, M.; Lundin, R. E.; Le, P. H. Proc.
Natl. Acad. Sci. USA 1988, 85, 5759.
0. Seo, S.; Uomori, A.; Yoshimura, Y.; Takeda, K.; Seto, H.;
3
Acknowledgements
Ebizuka, Y.; Noguchi, H.; Sankawa, U. J. Chem. Soc. Perkin
Trans. 1 1988, 2407.
This work is supported by the Welch Foundation
(D1276). We thank Wenxu Zhou for his e€orts to
determine the molecular weight of the SMT.
3
1. Nes, W. D.; Wong, R. Y.; Benson, M.; Landrey, J. R.;
Nes, W. R. Proc. Natl. Acad. Sci. USA 1984, 81, 5896.
2. Nes, W. D.; Wong, R. Y.; Benson, R. Y.; Akihisa, T. J.
Chem. Soc. Chem. Commun. 1991, 1272.
3. Nes, W. D.; Janssen, G. G.; Crumley, F. G.; Kalinoska,
M.; Akihisa, T. Arch. Biochem. Biophys 1993, 300, 724.
4. Nes, W. R.; Sekula, B. C.; Nes, W. D.; Adler, J. H. J. Biol.
3
3
References and Notes
3
1. Nes, W. R.; McKean, M. L. Biochemistry of Steroids and
Other Isopentenoids; University Park Press: Baltimore, 1977;
Chapters 9 and 10.
2. Nes, W. D.; Norton, R. A.; Crumley, F. G.; Madigan, S. J.;
Katz, E. R. Proc. Natl. Acad. Sci. 1990, 87, 7565.
Chem. 1978, 253, 6218.
35. Julia, M.; Marazanao, C. Tetrahedron 1985, 41, 3717.
36. Cornforth, J. W. Angew. Chem. Int. Engl. Ed. 1968, 7,
903.
37. Akhtar, M.; Jones, C. Tetrahedron 1978, 34, 813.
38. Lederer, E. Quart. Rev. Chem. Soc. Lond 1969, 23, 453.
39. Rahier, A.; Genot, J.; Schuber, F.; Benveniste, P.; Narula,
A. S. J. Biol. Chem. 1984, 259, 15215.
40. Parker, S. R.; Nes, W. D. Am. Chem. Soc. Symp. Ser 1992,
497, 110.
3. Bloch, K. E. CRC Crit. Rev. Biochem. 1983, 14, 47.
4. Hartman, M. Trends Plant Sci. 1998, 3, 170.
5. Goad, L. J.; Lenton, F. F.; Knapp, F. F.; Goodwin, T. W.
Lipids 1974, 9, 582.
. Nes, W. D.; McCourt, B. S.; Zhou, W.; Ma, J.; Marshall, J.
A.; Peek, L.; Brennan, M. Arch. Biochem. Biophys 1998, 353,
97.
6
41. Guo, D.; Jia, Z.; Nes, W. D. J. Am. Chem. Soc. 1996, 118,
8507.
2

9
36
A. T. Mangla, W. D. Nes / Bioorg. Med. Chem. 8 (2000) 925±936
4
A. L.; Lopez, M.; Li, H.; He, L. J. Org. Chem. 1999, 64, 1535.
2. Nes, W. D.; McCourt, B. S.; Marshall, J. A.; Ma, J.; Dennis, 53. Rahman, M. D.; Pascal, R. A., Jr. J. Biol. Chem. 1990,
265, 4989.
4
3. Nes, W. D.; Marshall, J. A.; Zhou, W.; He, L.; Dennis, A.
L. Tetrahedron Lett. 1998, 39, 8575.
4. Wokciechowski, Z. A.; Goad, L. J.; Goodwin, T. W. Bio-
chem. J. 1973, 136, 405.
54. Chung, S. K.; Ryoo, C. H.; Yang, H. W.; Shim, J-Y.;
Kang, M. G.; Lee, K. W.; Kang, H. I. Tetrahedron 1998, 54,
5899.
55. Acuna-Johnson, A. P.; Oehlschlager, A. C.; Pierce, A. M.;
Pierce, H. D., Jr; Czyzewska, E. K. Bioorg. Med. Chem. 1997,
5, 821.
56. Urbina, J. O.; Visbal, G.; Contreras, L.; McLaughlin, G.;
Decampo, R. Antimicrob. Agents Chemother. 1997, 41, 1428.
57. Rosser, R.; Faulkner, M. J. Org. Chem. 1984, 49, 5157.
58. Ator, M. A.; Schmidt, S. J.; Adams, J. L.; Dolle, R. E.
Biochemistry 1989, 28, 9633.
59. Nes, W. D.; He, L.; Mangla, A. T. Bioorg. Med. Chem.
Lett. 1998, 8, 3449.
60. Marshall, J. A.; Nes, W. D. Bioorg. Med. Chem. Lett.
1999, 9, 1533.
61. Schachterle, G. R.; Pollack, L. R. Anal. Biochem. 1973, 51,
654.
62. Nes, W. D.; Koike, K.; Jia, Z.; Sakamoto, Y.; Satou, T.;
Nakaido, T.; Grin, J. F. J. Am. Chem. Soc. 1998, 120, 5970.
4
4
1
4
5. Zhou, W-X. Master Thesis, Texas Tech University, August
999.
6. Diener, A.; Li, H.; Zhou, W-X.; Nes, W. D.; Fink, G.,
manuscript submitted for publication.
7. Yagi, T.; Morisaki, M.; Kushiro, T.; Yoshida, H.; Fuji-
moto, Y. Phytochemistry 1996, 41, 1057.
8. Mihailovic, M. M. PhD Dissertation, Eidgenoss Tech.
Hochsh., Zurich, Switzerland, 1984.
4
4
4
5
9. Arigoni, D. Ciba Found Symp. 1978, 60, 243.
0. Nes, W. D.; Janssen, G. G.; Bergenstrahle, A. J. Biol.
Chem. 1991, 266, 15202.
1. Oehlschlager, A. C.; Angus, R. H.; Pierce, A. M.; Pierce,
H. D. Jr; Srinivasan, R. Biochemistry 1984, 23, 3582.
2. Venkatramesh, M.; Guo, D.; Jia, Z.; Nes, W. D. Biochim.
Biophys. Acta 1996, 1299, 313.
5
5