Sterol methyl transferase. Evidence for successive C-methyl transfer reactions generating Δ24(28)- and Δ25(27)-olefins by a single plant enzyme

Article abstract of DOI:10.1016/S0040-4039(02)01550-2

The native SMT from Glycine max (soybean) expressed in Escherichia coli cells catalyzes the step-wise conversion of cycloartenol to 24(28)-methylene cycloartanol to a mixture of stereochemically related Δ²⁴⁽²⁸⁾Z-ethylidene-Δ²⁴⁽²⁸⁾E-ethylidene- and Δ²⁵⁽²⁷⁾-24β-ethylcyclosterols. [27-¹³C]Cycloartenol was tested as a substrate to determine the C-25 configuration of the Δ²⁴⁽²⁸⁾-olefin formed during the first C₁-transfer reaction. This substrate yielded (25-R)-[27-¹³C]24(28)-methylenecycloartanol. These results are entirely consistent with the observed stereochemistries of pathway specific 24-alkyl sterols found in plants and additionally revealed for the first time that a SMT can sequentially C-methylate the Δ²⁴-bond to generate multiple olefins.

Full text of DOI:10.1016/S0040-4039(02)01550-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7017–7021
Sterol methyl transferase. Evidence for successive C-methyl
transfer reactions generating D24(28)- and D25(27)-olefins by a
single plant enzyme
Allen L. Dennis and W. David Nes*
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
Received 23 July 2002; accepted 26 July 2002
Abstract—The native SMT from Glycine max (soybean) expressed in Escherichia coli cells catalyzes the step-wise conversion of
cycloartenol to 24(28)-methylene cycloartanol to a mixture of stereochemically related D24(28)Z-ethylidene-D24(28)E-ethylidene- and
D
25(27)-24b-ethylcyclosterols. [27-13C]Cycloartenol was tested as a substrate to determine the C-25 configuration of the D24(28)-olefin
formed during the first C1-transfer reaction. This substrate yielded (25-R)-[27-13C]24(28)-methylenecycloartanol. These results are
entirely consistent with the observed stereochemistries of pathway specific 24-alkyl sterols found in plants and additionally
revealed for the first time that a SMT can sequentially C-methylate the D24-bond to generate multiple olefins. © 2002 Elsevier
Science Ltd. All rights reserved.
Sterol methyl transferase (SMT) catalyzes the introduc-
advanced plants of clerosterol
5
(24b-ethyl) is
tion of C-28 and C-29 at C-24 and formation of D24(28)
-
considered to proceed by pathway specific routes as a
and D25(27)-olefin side chains giving rise to 24-alkyl
sterol diversity.1 The crucial role of these enzymes is to
generate phytosterols that serve as membrane inserts
and plant hormones.2 The biosynthesis in more-
advanced plants of sitosterol 8 (24a-ethyl) and in less-
result of differences in plant evolution (Scheme 1).1
Differential inhibition studies and analyses of the struc-
tural requirements for substrate recognition by plant
SMT3 as well as a recent investigation of the cloning,
Scheme 1. Hypothetical pathway in phytosterol synthesis.
Keywords: stereochemistry; phytosterol; sitosterol; cycloartenol; sterol; methyl transferase; soybean.
* Corresponding author. Tel.: (806)-742-1673; fax: (806)-742-0135; e-mail: wdavid.nes@ttu.edu
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01550-2

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A. L. Dennis, W. D. Nes / Tetrahedron Letters 43 (2002) 7017–7021
sequencing and functional expression of cDNA encod-
ing for SMT,4 have led several investigators to suggest
that a single enzyme catalyzes cycloartenol 1 to a
monoalkylated D24(28)-sterol side chain.4 Alternatively,
the doubly alkylated sterol side chain with specifically
the Z-ethylidene geometry 6 is thought to be generated
by a different enzyme species that fails to accept 24(28)-
methylenecycloartanol 2.5 However, since no plant
SMT has been purified to date, we hypothesized a
single plant SMT can catalyze both C-methylations by
the same reaction mechanism.3b Indeed, a comparison
of protein sequences deduced from the cDNAs for
fungal and plant SMTs (Fig. 1)4d revealed a highly
conserved region rich in aromatic amino acids, referred
to as Region I, containing a signature motif YEXGWX
not present in other AdoMet-dependent methyl trans-
ferases. As each of these plant enzymes uses
cycloartenol in similar fashion to fungal SMT which
catalyzes zymosterol, the aromatic-rich domain of
Region I is proposed to be involved in substrate bind-
ing, and possibly product formation, by stabilizing
intermediate carbenium ions generated during sterol
C-methylation.3
recombinant SMT. Incubation with lanosterol gener-
ated 24(28)-methylene lanosterol. The investigators
concluded the soybean SMT catalyzes only the first
C1-transfer reaction. We transferred the SMT cDNA
from the pSERT to the T7-based high-level expression
system, pET23a plasmid, by introducing a restriction
site NdeI at the 5%-end and a BamHI site at the 3%-end.9
SMT cDNA localized in pET23a vector was trans-
formed into BL21(DE3) cells at 37°C and expressed by
addition of IPTG to the culture medium.9 Cell were
lysed using a sonifier, and the resulting native soybean
SMT was resuspended in 50 mM Tris–HCl, 2 mM
MgCl2, 2 mM b-mercaptoethanol and 5% glycerol (v/v)
at pH 7.5.9 Activity assays using cell-free
homogenates7d were performed overnight with saturat-
ing amounts of AdoMet and cycloartenol or 24(28)-
methylene cycloartanol at 35°C and products of
1
incubation were analyzed by GC–MS and H NMR.
Total sterols were recovered from the reactions essen-
tially as described previously.9 Reactions generated at
least 300 mg of total product and the sterol mixture was
purified to give individual compounds by HPLC using
TSK gel C18-columns.10 GC–MS analysis of the
enzyme-generated product from the cycloartenol incu-
bation showed the product of C-methylation to corre-
spond to an authentic specimen of 24(28)-
methylenecycloartanol (Fig. 2; panel A).10 By contrast,
when 24(28)-methylenecycloartanol was incubated sep-
arately with the SMT enzyme three peaks were
observed with base-line resolution in GC. The mass
spectra of these compounds exhibited similar fragmen-
tation patterns (M+ 454 amu) corresponding to 24-ethyl
sterol type lanostanoids, which we and others have
isolated from fungi11 (Fig. 2; panel B). HPLC chro-
matographic purification of the crude non-saponifiable
lipid fraction of a preparative-scale incubation of
24(28)-methylenecycloartanol with SMT gave substrate
and three novel products 9, 10, and 11. The chromato-
graphic and spectral data of the C-methylated cyclos-
terols generated by soybean SMT during the second
C1-transfer were as follows: 9, GLC (retention time
relative to cholesterol–RRTc on DB5-capillary
columns,10 2.39); MS (m/z 454 (M+), 439, 421, 393, 355,
Concerning the stereochemical features of these trans-
formations, there is evidence that the direction of trans-
fer of the C-methyl group from AdoMet proceeds by
alkylation from the re (a)-face6 or the si (b)-face7 of
D24. To date, nothing has been reported about the
ability of a native SMT enzyme to possess variant
activities that include substrate channeling to form
multiple olefins.8 We now report investigations designed
to identify enzyme-generated product stereochemistries
and the stereochemistry of hydrogen migration from
C-24 to C-25 in the biosynthesis of 24(28)-methylenecy-
cloartanol using soluble recombinant soybean (Glycine
max) SMT which allows the number of SMTs and the
C-methylation mechanism involved with 24-ethyl sterol
and multiple olefin formation to be established.
Shi et al.4c isolated the soybean SMT cDNA and pro-
vided us their plasmid pSERT carrying FLAG-epitope
tagged SMT1 cDNA that generates a fusion protein. In
the Shi et al. study, lanosterol was assayed using a
cell-free homogenate of Escherichia coli expressing the
1
341, 323; 500 MHz H NMR l ppm, 0.538 (H-18, s),
Figure 1. Partial sequence alignment and predicted Mr (kDa) and pI of 16 SMTs of plant and fungal origin (A).4d Yellow colored
indicates a conserved residue and boxed area indicates signature moitf. Start residue in parentheses. Phylogenetic tree illustrating
SMT relatedness was generated by Neighbor Joining Method embedded in the Vector NTI Suite 6.0 software package (Infomax)
(B).

A. L. Dennis, W. D. Nes / Tetrahedron Letters 43 (2002) 7017–7021
7019
Figure 2. GC trace of (A) cycloartenol incubation with recombinant SMT and (B) 24(28)-methylenecycloartanol incubation with
recombinant SMT. Peaks are as follow: 1, cycloartenol; 2, 24(28)-methylenecycloartanol; 9, 24b-ethyl-25(27)-dehydrocycloartanol;
10, 24(28)E-24-ethylidene cycloartanol and; 11, 24(28)Z-24-ethylidene cycloartanol.
0.556 and 0.333 (H-19, d, 4.2=endo, d, 4.2=exo),
0.898 (H-21, d, 7.0), 1.568 (H-26, s), 0.804 (H-29, t),
0.984 (H-30, s), 3.126 (H-3, dd, 11.7, 4.4), 4.731 and
4.647 (H-27, s, s); 10, GLC (RRTc, 2.43); MS (m/z 454
endo, d, 4.2=exo), 0.899 (H-21, d, 6.4), 1.024 (H-26,
dd, 5.2, 6.8), 1.017 (H-27, dd, 1.25.5, 6.8), 0.968 (H-30,
s), 0.811 (H-31, s), 0.901 (H-32, s), 3.126 (H-3, m),
4.716, 4.665 (H-28, d, 1.1; d, 1.1); 13C NMR (terminal
side chain carbons) 125 MHz l ppm C-24 (156.94),
C-25 (33.65), C-26 (21.86), C-27 (21.99), C-28 (105.90).
As shown in Fig. 3, the signal enhanced in the 13C
NMR spectrum of enzyme-generated 13C-labeled 2 cor-
responds to C-27 and reveals this carbon atom contains
the pro-R methyl group.7d
1
(M+), 439, 421, 393, 355, 341, 323); H NMR, 0.538
(H-18, s), 0.556 and 0.333 (H-19, d, 4.2=endo, d,
4.2=exo), 0.896 (H-21, d, 6.4), 2.358 (H-25, sept, 7.0),
0.984 (6Hs, H-26, H-27, d, 6.6), 1.570 (H-29, d, 6.7),
0.984 (H-30, s), 0.811 (H-31, s), 0.901 (H-32, s), 3.126
(H-3, m), 5.185 (H-25, q, 6.7,), 5.181 (H-28, q, 6.8; 11,
GLC (RRTc, 2.47); MS (m/z 454 (M+), 439, 421, 393,
1
355, 341, 323); H NMR, 0.538 (H-18, s), 0.556 and
Based on steady-state kinetic analysis, 24(28)-
methylenecycloartanol (Kmapp=12 mM/Vmaxapp=14
pmol/min/mg) is a worse substrate to cycloartenol
(Kmapp=20 mM/Vmaxapp=243 pmol/min/mg) for soy-
bean SMT catalysis. As much as 10 mM cycloartenol
added to the incubation containing 24(28)-methylenecy-
cloartanol prevents 24-ethyl sterol product formation,
0.333 (H-19, d, 4.2=endo, d, 4.2=exo), 896 (H-21, d,
6.4), 2.834 (H-25, sept, 7.0), 0.984 (6Hs, H-26, H-27, d,
6.6), 1.584 (H-29, d, 6.7), 0.984 (H-30, s), 0.811 (H-31,
s), 0.901 (H-32, s), 3.126 (H-3, m); 5.110 (H-28, q, 6.8).
The stereochemistry at C-24 as b-oriented in 9 is
7e,12
demonstrated by the signal for C-29
and the E/Z-
geometry at C-24 for 10 and 11 is demonstrated by the
signals for C-25, C-28 and C-29.13 Full 1H and 13C
NMR assignments of cycloartenol are reported.12c
From these data the second C1-transfer in soybean
sterol C-methylation may be concluded to occur from
the si-face of the D24-bond.
The stereochemistry of hydrogen migration from C-24
of cycloartenol to C-25 in the biosynthesis of 24(28)-
methylene cycloartanol is problematic since the product
of C-methylation is an exomethylene structure. There-
fore, to unambiguously establish the product 25R-stere-
ochemistry in 2 soybean SMT was incubated with
[27-13C]1. The product possessed the following chro-
matographic and spectral cycloartenol characteristics:
GLC (RRTc, 1.87); MS (m/z 441 (M+)), 426, 408, 380,
354, 301 (cyclopropane ring fragment); 1H NMR l
ppm, 0.968 (H-18, s), 0.556 and 0.333 (H-19, d, 4.2=
Figure 3. Partial 13C NMR spectrum of (25R)-[27-13C]
24(28)methylenecycloartanol recovered from soybean SMT
incubated with [27-13C].

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A. L. Dennis, W. D. Nes / Tetrahedron Letters 43 (2002) 7017–7021
H
CH2
CH2
H
13C
13C
H
13C
SMT
HO
AdoMet
HO
2
1
HO
SMT
AdoMet
path a
path b
CH3
H
H
C
a
b
HO
HO
HO
H
H
c
CH2
11
10
9
HO
path c
Scheme 2. C-Methylation pathway in phytosterol synthesis.
suggesting that under normal physiological conditions
the second C1-transfer is limited. Recently, we charac-
terized the kinetic parameters of a soybean SMT
tion of three olefins during the second C1-transfer reac-
tion is presumably made possible by the relaxed control
over substrate and intermediate conformations in the
active site as a result of the increased steric bulk derived
from the 24-ethyl sterol intermediate. Notably, the
deprotonations leading to the formation of 9, 10, and
11 involve substrate channeling along different reaction
coordinates (Scheme 2) suggesting the second C1-trans-
fer reaction is not necessarily concerted.
obtained
from
shoots
which
binds
24(28)-
methylenelophenol preferentially to cycloartenol and
24(28)-methylenecycloartanol was not a substrate for
the enzyme.3a In addition, soybean plants apparently
do not normally synthesize D24(28)(E) or 24b-ethyl
D
25(27)-sterol side chains.14 These findings suggest bind-
ing order of substrate can contribute to product diver-
sity and that at least two SMT isoforms exist in
soybean to control substrate flux to sitosterol (24a-ethyl
cholesterol).
Acknowledgements
The general picture which emerges from these and
related studies is one in which the pro-Z methyl group
on cycloartenol corresponding to C-27 is transformed
into the pro-R-methyl group at C-25 on 24(28)-
methylenecycloartanol by migration of the hydrogen
atom at C-24 to C-25 from the re-face of the substrate
double bond of the acceptor molecule (Scheme 2). The
first C1-transfer reaction is proposed to proceed via a
non-covalent pathway whereby methyl addition to the
si face of D24-and deprotonation of C-28 gives rise to a
nucleophilic rearrangement in which H-24 migrates to
C-25 on the opposite face of the substrate double bond
in concert with the initial ionization, consistent with
yeast SMT action.3a,7a In the case of the second C1-
transfer reaction, the C-methylation reaction can also
proceed from the si-face of the D24-bond. The forma-
This work was supported by the NSF (MCB 0115401
and the Welch Foundation (D-1276). We thank Dr.
Haoxia Li for generating the SMT cDNA construct
used in this investigation and for technical assistance in
the preliminary experiments.
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