Directed Evolution to Generate Cycloartenol Synthase Mutants that Produce Lanosterol

Article abstract of DOI:10.1021/ol0257225

(Matrix Presented) Cycloartenol synthase converts oxidosqualene to cycloartenol, a pentacyclic isomer of the animal and fungal sterol precursor lanosterol. We used directed evolution to find cycloartenol synthase residues that affect cyclopropyl ring formation, selecting randomly generated cycloartenol synthase mutants for their ability to genetically complement a yeast strain lacking lanosterol synthase. To increase the likelihood of finding novel mutations, the little-studied Dictyostelium discoideum cycloartenol synthase was used for the mutagenesis. Several catalytically important residues were identified.

Full text of DOI:10.1021/ol0257225

ORGANIC
LETTERS
2002
Vol. 4, No. 8
1395-1398
Directed Evolution To Generate
Cycloartenol Synthase Mutants that
Produce Lanosterol
Michelle M. Meyer, Ran Xu, and Seiichi P. T. Matsuda*
Department of Chemistry and Department of Biochemistry and Cell Biology,
Rice UniVersity, 6100 South Main Street, Houston, Texas 77005
matsuda@rice.edu
Received February 14, 2002
ABSTRACT
Cycloartenol synthase converts oxidosqualene to cycloartenol, a pentacyclic isomer of the animal and fungal sterol precursor lanosterol. We
used directed evolution to find cycloartenol synthase residues that affect cyclopropyl ring formation, selecting randomly generated cycloartenol
synthase mutants for their ability to genetically complement a yeast strain lacking lanosterol synthase. To increase the likelihood of finding
novel mutations, the little-studied Dictyostelium discoideum cycloartenol synthase was used for the mutagenesis. Several catalytically important
residues were identified.
Oxidosqualene cyclases convert oxidosqualene (Scheme 1,
1) through carbocationic intermediates to nearly 100 different
cyclic triterpene alcohols.1 These enzymes generate triter-
penoid diversity by controlling which proton is abstracted
from a carbocationic intermediate. For example, both lano-
sterol synthase and cycloartenol synthase cyclize oxi-
dosqualene to the protosteryl cation (2) and then promote
rearrangement to the lanosteryl 8-cation (3). Lanosterol
synthase abstracts the proton originating at C-9 to form
lanosterol (Scheme 1, 4), either directly from C-9 or after a
hydride shift from C-9 to C-8. Cycloartenol (5) biosynthesis
requires a hydride shift from C-9 to C-8, followed by
cyclopropyl ring formation and deprotonation from C-19
(Scheme 1).
catalytic difference between cycloartenol synthase and lano-
sterol synthase. A selection for randomly generated AtCAS1
mutants that genetically complement a yeast lanosterol
synthase deletion strain uncovered an Ile481Val mutant that
converts oxidosqualene to a mixture of cycloartenol, lano-
sterol, and parkeol (6) (55:24:21).3 The strict differential
conservation at this position (Ile is conserved in cycloartenol
synthases and Val is conserved in lanosterol synthases, Figure
1) shows that the residue at this position contributes to the
catalytic difference between native cycloartenol synthase and
lanosterol synthase. At four other positions (AtCAS1 Tyr410,
Gly488, Phe717, and Met731) cycloartenol synthases strictly
conserved one residue and the known lanosterol synthases
conserved another. We generated AtCAS1 derivatives with
these residues mutated to their lanosterol synthase counter-
We previously identified two Arabidopsis thaliana cy-
cloartenol synthase2 (AtCAS1) residues that contribute to the
(2) Corey, E. J.; Matsuda, S. P. T.; Bartel, B. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 11628-11632.
(1) (a) Abe, I.; Rohmer, M.; Prestwich, G. D. Chem. ReV. 1993, 93,
2189-2206. (b) Matsuda, S. P. T. In Biochemical Principles and Mech-
anisms of Biosynthesis and Biodegradation of Polymers; Steinbu¨chel, A.,
Ed.; Wiley-VCH: Weinheim, 1998; pp 300-307.
(3) (a) Hart, E A.; Hua, L.; Darr, L. B.; Wilson, W. K.; Pang, J.; Matsuda,
S. P. T. J. Am. Chem. Soc. 1999, 121, 9887-9888. (b) Matsuda, S. P. T.;
Darr, L. B.; Hart, E. A.; Herrera, J. B. R.; McCann, K. E.; Meyer, M. M.;
Pang, J.; Schepmann, H. G. Org. Lett. 2000, 2, 2261-2263.
10.1021/ol0257225 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/19/2002

Scheme 1. Cyclization and Rearrangement of Oxidosqualene (1) to Cyclic Triterpene Alcohols; Lanosterol (4) Accessible in Principle
by Either Deprotonating the C8 or C9 Cation or a Bridged Structure
parts to identify those that control product structure. Only
the Tyr410Thr mutant had an altered product profile, forming
lanosterol, 9â-lanosta-7,24-dien-3â-ol (Scheme 1, 7), and
parkeol (65:33:2).4 This mutation is cooperative with the
Ile481Val mutation; the Ile481Val Tyr410Thr double mutant
biosynthesizes lanosterol more efficiently (75:24:0.6),4 but
deprotonation from C7 and C11 also occurs.
Because all positions with strict differential conservation
had been examined, we revisited directed evolution to
identify other catalytically relevant positions. To increase
the chance of obtaining mutations different from the Ile481Val
found in the initial selection ofAtCAS1 mutants, we used
the Dictyostelium discoideum ortholog (DdCAS1). Randomly
mutated libraries5 of DdCAS1 were transformed into the
yeast lanosterol synthase deletion mutant SMY8.6 Yeast
transformants, marked by uracil prototrophy, were induced
and selected on plates containing galactose and lacking
ergosterol.7 The mutant enzymes that acquired lanosterol
biosynthetic ability were expected to allow SMY8 to grow
in the absence of ergosterol.
Nonsaponifiable lipid was obtained from strains that grew
without exogenous sterol. Yeast metabolizes lanosterol
rapidly to ergosterol, so the presence of ergosterol (evidenced
by GC-MS) in each positive strain confirmed its ability to
biosynthesize lanosterol. SMY8 was retransformed with each
mutant construct, and the transformants were rescreened by
the same complementation assay to ensure that the plasmids
provided the sterol independence. Plasmids recovered from
positive strains were sequenced, and each had a single
mutation.
Eighteen strongly complementing plasmids were isolated
from 10,000 transformants. Each had one of four muta-
Figure 1. Conservation pattern of the mutated residues. Ile481 (1) is differentially conserved as Ile in cycloartenol synthases (CAS1) and
Val in lanosterol synthases (ERG7). Phe472 (b) and Tyr532 (*) are conserved in the known cycloartenol synthases and lanosterol synthases,
and Ser482 ([) is nearly conserved. Cycloartenol synthases are from A. thaliana (At),2 Pisum satiVum (Ps),12a Panax ginseng (Pg),12b Luffa
cylindrica (Lc),12c Glycyrrhiza glabra (Gg),12d and D. discoideum (Dd).12e Lanosterol synthases are from S. cereVisiae (Sc),13c,d Candida
albicans (Ca),13a,b Schizosaccharomyces pombe (Sp),6 Rattus norVegicus (Rn),13e,f Homo sapiens (Hs),13g,h Trypanosoma brucei (Tb),13i and
Trypanosoma cruzi (Tc).13j
1396
Org. Lett., Vol. 4, No. 8, 2002

tions: DdCAS1 Tyr363Cys, Phe424Ser, Ser434Pro, and
Tyr481His. Phe424Ser arose only once, but the others were
observed repeatedly. Because all previous analyses of
cycloartenol synthase mutants had been done in AtCAS1,
we maintained consistency by generating the four corre-
sponding AtCAS1 mutants using site-specific mutagenesis:
Tyr410Cys, Phe472Ser, Ser482Pro, and Tyr532His. The
AtCAS1 Tyr410Cys and Tyr532His mutants genetically
complemented the sterol auxotrophy in SMY8, allowing
growth comparable to the wild-type lanosterol synthase
cDNA expressed identically in SMY8 (colonies formed after
2 days). However, AtCAS1 Phe472Ser complemented less
efficiently (colonies formed in 7 days), and yeast that were
dependent on AtCAS1 Ser482Pro for sterol did not generate
colonies after 2 weeks. At all four mutated positions, AtCAS1
and DdCAS1 conserve the same residue, and AtCAS1 could
acquire single nucleotide mutations parallel to those observed
in DdCAS1. The strongly complementing AtCAS1 Tyr410Cys
and Tyr532His mutants did not appear in an earlier screen
for spontaneously occurring mutations.3a This probably
reflects the higher mutagenesis rate in the current study rather
than differences between the two genes.
been solved to 2.0 Å resolution, and Tyr410 corresponds to
the active-site SHC Ser307 residue.11 The AtCAS1 Tyr410Cys
mutant makes significant amounts of lanosterol because the
substituted Cys sterically and electronically resembles the
Thr that is important in animal and fungal lanosterol
synthases.
AtCAS1 Tyr532His does not biosynthesize cycloartenol
but a mixture of 45% lanosterol, 31% parkeol, and 24%
achilleol A. AtCAS1 Tyr532His is less efficient than native
AtCAS1, generating ∼5 mg/L culture (∼10% the yield from
native enzyme). Tyr532 is strictly conserved in both cy-
cloartenol synthase2,12 and lanosterol synthase6,13 and there-
fore probably does not play a role specific to cyclopropyl
ring formation. Instead, the formation of substantial amounts
of the monocyclic achilleol A in the Tyr532His mutant
suggests that Tyr532 may facilitate cyclization to the
tetracyclic cation. Interestingly, Trp is conserved at the
corresponding position in enzymes that initially cyclize to
the dammarenyl cation, and the Tyr/Trp dichotomy may
influence not only the number of rings that form but also
the stereochemistry of intermediate tetracyclic cations.
AtCAS1Tyr532 corresponds to the active site SHC Tyr420
residue, and mutating SHC Tyr420 also generated partially
cyclized products.14 The AtCAS1 Tyr532His mutation neither
introduces (as does the Ile481Val mutation) nor structurally
mimics (as does the Tyr410Cys mutation) a lanosterol
synthase residue yet allows lanosterol biosynthesis. Depro-
tonation to lanosterol and parkeol rather than ring-closure
to cycloartenol may be an indirect effect. Tyr532His is
compatible with lanosterol biosynthesis, but the absence of
this substitution in native lanosterol synthases suggests that
His may compromise accurate, efficient lanosterol produc-
tion.
The AtCAS1 Tyr410Cys mutant converts oxidosqualene
to lanosterol, 9â-lanosta-7,24-dien-3â-ol, and achilleol A (8)
(75:24:1).8 This product composition is readily rationalized
in light of previous experiments. Tyr410 is conserved in
cycloartenol synthases and corresponds to a Thr that is
conserved in animal and fungal lanosterol synthases. This
position was shown previously to be catalytically important
in both cycloartenol synthase2,12 and lanosterol synthase.6,13
The AtCAS1 Tyr410Thr mutant does not produce cy-
cloartenol but instead lanosterol, 9â-lanosta-7,24-dien-3â-
ol, and parkeol (65:33:2).4 Mutating the corresponding
Thr384 residue in yeast lanosterol synthase to the Tyr
conserved in cycloartenol synthase compromised selectivity.
Although lanosterol remained the major product, parkeol and
lanost-24-ene-3â,9R-diol appeared as byproducts.9 Neither
cycloartenol synthase nor lanosterol synthase has been
structurally characterized, but the structure of a related
enzyme known as squalene-hopene cyclase (SHC)10 has
Like Tyr532, Phe472 is strictly conserved in both cy-
cloartenol and lanosterol synthases and consequently does
not contribute to the catalytic difference between native
lanosterol synthases and cycloartenol synthases. AtCAS1
(11) (a) Wendt, K. U.; Poralla, K.; Schulz, G. E. Science 1997, 277,
1811-1815. (b) Wendt, K. U.; Lenhart, A.; Schulz, G. E. J. Mol. Biol.
1999, 286, 175-187.
(12) (a) Morita, M.; Shibuya, M.; Lee, M.-S.; Sankawa, U.; Ebizuka, Y.
Biol. Pharm. Bull. 1997, 20, 770-775. (b) Kushiro, T.; Shibuya, M.;
Ebizuka, Y. Eur. J. Biochem. 1998, 256, 238-244. (c) Hayashi, H.; Hiraoka,
N.; Ikeshiro, Y.; Yazaki, K.; Tanaka, S.; Kushiro, T.; Shibuya, M.; Ebizuka,
Y. Plant Physiol. 1999, 121, 1384. (d) Hayashi, H.; Hiraoka, N.; Ikeshiro,
Y.; Kushiro, T.; Morita, M.; Shibuya, M.; Ebizuka, Y. Biol. Pharm. Bull.
2000, 23, 231-234. (e) Godzina, S. M.; Lovato, M. A.; Meyer, M. M.;
Foster, K. A.; Wilson, W. K.; Gu, W.; de Hostos, E. L.; Matsuda, S. P. T.
Lipids 2000, 36, 249-255.
(4) Herrera, J. B. R.; Wilson, W. K.; Matsuda, S. P. T. J. Am. Chem.
Soc. 2000, 122, 6765-6766.
(5) Mutant libraries were generated by propagation in the mutagenic E.
coli strain XL1-Red (Stratagene) according to the manufacturer’s instruc-
tions.
(6) Corey, E. J.; Matsuda, S. P. T.; Baker, C. H.; Ting, A. Y.; Cheng,
H. Biochem. Biophys. Res. Commun. 1996, 219, 327-331.
(7) Lovato, M. A.; Hart, E. A.; Segura, M. J. R.; Giner, J.-L.; Matsuda,
S. P. T. J. Biol. Chem. 2000, 275, 13394-13397.
(8) Each enzyme was assayed in vitro using synthetic oxidosqualene and
protein expressed from the galactose-inducible vector pRS305GAL in the
yeast strain LHY4 as described previously.3 Catalytically efficient mutants
typically provided >50 mg product from a crude yeast lysate generated
from 1 L of culture. Because each polycyclic triterpene has multiple
distinctive methyl signals, known triterpene alcohols were readily identified
by 1H NMR of a partially purified mixture. Acetate derivatives were
prepared, and GC-MS analyses with comparison to authentic samples were
performed to ensure that no oxidosqualene cyclase products were overlooked
in the NMR. GC-FID was used for quantitation.
(13) (a) Buntel, C. J.; Griffin, J. H. J. Am. Chem. Soc. 1992, 114, 9711-
9713. (b) Roessner, C. A.; Min, C.; Hardin, S. H.; Harris-Haller, L. W.;
McCollum, J. C.; Scott, A. I. Gene 1993, 127, 149-150. (c) Corey, E. J.;
Matsuda, S. P. T.; Bartel, B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2211-
2215. (d) Shi, Z.; Buntel, C. J.; Griffin, J. H. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 7370-7374. (e) Kusano, M.; Shibuya, M.; Sankawa, U.; Ebizuka,
Y. Biol. Pharm. Bull. 1995, 18, 195-197. (f) Abe, I.; Prestwich, G. D.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9274-9278. (g) Baker, C. H.;
Matsuda, S. P. T.; Liu, D. R.; Corey, E. J. Biochem. Biophys. Res. Commun.
1995, 213, 154-160. (h) Sung, C.-K.; Shibuya, M.; Sankawa, U.; Ebizuka,
Y. Biol. Pharm. Bull. 1995, 18, 1459-1461. (i) Buckner, F. S.; Ngyuen,
L. N.; Joubert, B. M.; Matsuda, S. P. T. Mol. Biochem. Parasitol. 2000,
110, 399-403. (j) Joubert, B. M.; Buckner, F. S.; Matsuda, S. P. T. Org.
Lett. 2001, 3, 1957-1960. (k) Abe, I.; Naito, K.; Takagi, Y.; Noguchi, H.
Biochim. Biophy. Acta 2001, 1522, 67-73.
(9) Meyer, M. M.; Segura, M. J. R.; Wilson, W. K.; Matsuda, S. P. T.
Angew. Chem. 2000, 112, 4256-4258. Angew. Chem., Int. Ed. 2000, 39,
4090-4092.
(10) Ochs, D.; Kaletta, C.; Entian, K.-D.; Beck-Sickinger, A.; Poralla,
K. J. Bacteriol. 1992, 174, 298-302.
(14) Pale-Grosdemange, C.; Merkofer, T.; Rohmer, M.; Poralla, K.
Tetrahedron Lett. 1999, 40, 6009-6012.
Org. Lett., Vol. 4, No. 8, 2002
1397

Phe472 corresponds to the catalytically important active site
Phe365 residue in SHC.15 The AtCAS1 Phe472Ser mutant
has severe catalytic defects. Although in vitro reactions
provided insufficient material (<5 µg/L culture, <0.01% the
yield from native enzyme) to rigorously characterize the
products by 1H NMR,16 its ability to complement the
lanosterol synthase mutant implies that Phe472Ser promotes
low-level lanosterol biosynthesis.17 Enzymes with a Phe472Ser
probably would not be efficient enough to support competi-
tive growth in nature.
AtCAS1 Ser482 is strongly but incompletely conserved,
being present in all but the Saccharomyces13c,d and rat13e,f
lanosterol synthases (which have Ala substitutions). AtCAS1
Ser482 corresponds to the SHC active-site Val375 residue.
In vitro reactions did not provide any evidence of oxi-
dosqualene cyclization. This difference in behavior between
the DdCAS1 Ser434Pro mutant and the parallel AtCAS1
Ser482Pro mutant highlights the importance of maintaining
identical backgrounds when comparing the effects of muta-
tions.
These experiments show that random mutagenesis and
selection can uncover a variety of mutations that allow a
cycloartenol synthase to biosynthesize lanosterol and thereby
genetically complement a yeast lanosterol synthase mutant.
Each mutation maps to the active site. The substitutions
generated by directed evolution are absent in native lanosterol
synthases and are probably incompatible with efficient,
accurate lanosterol biosynthesis. Tyr410Cys mimics the Thr
in native lanosterol synthase, but Phe472Ser and Tyr532His
replace residues that are strictly conserved (or nearly so) in
both cycloartenol and lanosterol synthases. The latter two
mutations are perhaps best viewed as generating enzymes
that have broadened specificity and make lanosterol as one
of the products. The tetracyclization and rearrangement of
oxidosqualene to cation 3 appear to be more robust than the
ring closure from 3 to cycloartenol, and mutations in residues
with conservation patterns that seem inconsistent with a
specific role in cyclopropyl ring formation can disrupt
cyclopropyl ring formation while allowing tetracyclization.
Acknowledgment. We thank Jihai Pang for expert GC-
MS analyses and William K. Wilson for advice on NMR.
We thank Silvia Lodeiro, Kelly E. McCann, and Katherine
A. Walther for critical comments on the manuscript. This
research was funded by the NIH (AI41598) and the Robert
A. Welch Foundation (C-1323).
(15) Hoshino, T.; Sato, T. Chem. Commun. 1999, 1999, 2005-2006.
(16) 1H NMR is essential for reliable characterization because many
triterpene alcohols have similar chromatographic and fragmentation proper-
ties (lanosterol and its ∆-7 isomer are essentially indistinguishable by GC-
MS).
(17) Our experience with expressing lanosterol synthases indicates that
the yeast complementation assay is considerably more sensitive than GC
for identifying low levels of lanosterol biosynthesis.
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