Alteration of the substrate's prefolded conformation and cyclization stereochemistry of oxidosqualene-lanosterol cyclase of saccharomyces cerevisiae by substitution at Phenylalanine 699

Article abstract of DOI:10.1021/ol902694y

[Chemical equaction presented] The Saccharomyces cerevisiae ERG7 ^Phe699 mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17α-exocyclic unrearranged intermediate, and

Full text of DOI:10.1021/ol902694y

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ORGANIC
LETTERS
2010
Vol. 12, No. 3
500-503
Alteration of the Substrate’s Prefolded
Conformation and Cyclization
Stereochemistry of
Oxidosqualene-Lanosterol Cyclase of
Saccharomyces cerevisiae by
Substitution at Phenylalanine 699
Tung-Kung Wu,* Cheng-Hsiang Chang, Hao-Yu Wen, Yuan-Ting Liu,
Wen-Hsuan Li, Tsai-Ting Wang, and Wen-Shiang Shie
Department of Biological Science and Technology, National Chiao Tung UniVersity,
300, Hsin-Chu, Taiwan, Republic of China
tkwmll@mail.nctu.edu.tw
Received November 23, 2009
ABSTRACT
The Saccharomyces cerevisiae ERG7Phe699 mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated
tricyclic compounds, one tetracyclic 17r-exocyclic unrearranged intermediate, and two 17ꢀ-exocyclic truncated rearranged intermediates.
These results provided direct evidence for the importance of the residue in affecting mechanistic transitions between C-B-C and C-C-C
substrate conformation and between the 17r- and 17ꢀ-exocyclic side chain stereochemistry as well as in stabilizing the 6-6-5 tricyclic and
the protosteryl C-17 cations.
The advent of contemporary protein engineering techniques,
including mutation, recombination, and selection, has paved
the way to understand the structure-function-mechanism
relationships of enzyme catalysis and to tailor enzymes with
new catalytic activity and selectivity.1 Identification of
plasticity residues in target proteins with diverse product
profiles provides the starting point to understand how proteins
acquire novel or altered functions and how plasticity residues
contribute to the natural evolution process as well as allow
an efficient design methodology for new enzymes to be
formulated. Plasticity has been studied in a number of
enzymes and recent findings have shown a divergent strategy
for shaping cyclization of (3S)-2,3-oxidosqualene (1, OS),
wherein mutational steps alter the biosynthetic properties of
enzymes. Interestingly, the fascinating enzymes, the oxi-
dosqualene cyclases (OSC or ERG7), cyclize acyclic OS into
diverse polycyclic sterols and triterpenoids with two funda-
(2) (a) Abe, I.; Rohmer, M.; Prestwich, G. D. Chem. ReV. 1993, 93,
2189–2206. (b) Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R. Angew.
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S. P. T. Phytochemistry 2004, 65, 261–291. (d) Abe, I. Nat. Prod. Rep.
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2002, 6, 125–129. (b) Yoshikuni, Y.; Ferrin, T. E.; Keasling, J. D. Nature
2006, 440, 1078–1082. (c) Glasner, M. E.; Gerlt, J. A.; Babbitt, P. C. AdV.
Enzymol. Relat. Areas Mol. Biol. 2007, 75, 193–239.
10.1021/ol902694y  2010 American Chemical Society
Published on Web 01/07/2010

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mental biosynthetic routes, based either on chair-boat-chair
(C-B-C) or chair-chair-chair (C-C-C) prefolded sub-
strate conformation.2-8 In addition, cyclase-catalyzed reac-
tions can proceed in either an accurate or multifunctional
fashion to achieve catalytic perfection or to generate diverse
product profiles.3,5 The isolated product profiles reveal
the catalytic function of the given amino acids and support
the idea that subtle changes in individual residues can have
electronic effects and/or can sterically alter the active site
cavity structure to generate diverse product profiles. How-
ever, the functional residues involved in determining sub-
strate specificity for squalene/oxidosqualene, C-B-C/
C-C-C substrate conformation, and 17R/17ꢀ stereochemical
control of exocyclic side chain, remain unsolved.
To explore whether a single-site mutation in enzymes
possessing the cyclase structure is sufficient to alter substrate
conformation and protosteryl 17R/17ꢀ stereochemistry,
amino acids spatially near the substrate B/C/D ring positions
were targeted. We have previously characterized a functional
role of Saccharomyces cereVisiae ERG7F699T mutation in
influencing protosteryl C-17 cation stabilization.3h A more
complex interaction between Phe699 and prefolded substrate
conformation or cationic intermediates is expected and
provokes further investigation into the effects of substitution
of other amino acids on the catalytic activity and capacity
to traverse mechanistic barriers between C-B-C and
C-C-C substrate conformation and between 17R and 17ꢀ
stereochemistry of tetracyclic intermediates.
The Phe699 position of ERG7 was exchanged with other
amino acids using the QuikChange Site-Directed Mutagen-
esis Kit and transformed into the S. cereVisiae TKW14 and
YTL4 strains, as previously described.3h The genetic selec-
tion results showed that TKW14[pERG7F699X] site-saturated
mutants allowed for ergosterol-independent growth, with the
exception of Leu, Ile, His, Met, Pro, and the previously
reported Thr substitutions. Next, the product profiles gener-
ated by the ERG7 mutant enzymes were analyzed, using the
YTL4[pERG7F699X] strain, and characterized by GC-MS and
NMR (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC,
HMBC, and NOE) spectroscopies.3f
Different ERG7F699X mutants produced diverse product
profiles, ranging from none to as many as seven compounds
with molecular mass of 426 Da. Among them, four com-
pounds were indistinguishable from authentic lanosterol (2,
LA), protosta-13(17)-dien-3ꢀ-ol (3), (13RH)-isomalabarica-
14E,17E,21-dien-3ꢀ-ol (7), and (13RH)-isomalabarica-
(3) (a) Wu, T. K.; Chang, C. H.; Liu, Y. T.; Wang, T. T. Chem. Rec.
2008, 8, 302–325. (b) Wu, T. K.; Griffin, J. H. Biochemistry 2002, 41,
8238–8244. (c) Wu, T. K.; Chang, C. H. ChemBioChem 2004, 5, 1712–
1715. (d) Wu, T. K.; Liu, Y. T.; Chang, C. H. ChemBioChem 2005, 6,
1177–1181. (e) Wu, T. K.; Yu, M. T.; Liu, Y. T.; Chang, C. H.; Wang,
H. J.; Diau, E. W. G. Org. Lett. 2006, 8, 1319–1322. (f) Wu, T. K.; Liu,
Y. T.; Chang, C. H.; Yu, M. T.; Wang, H. J. J. Am. Chem. Soc. 2006, 128,
6414–6419. (g) Wu, T. K.; Liu, Y. T.; Chiu, F. H.; Chang, C. H. Org. Lett.
2006, 8, 4691–4694. (h) Wu, T. K.; Wen, H. Y.; Chang, C. H.; Liu, Y. T.
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Liu, Y. T. Org. Lett. 2008, 10, 4959–4962. (j) Wu, T. K.; Li, W.-H.; Chang,
C.-H.; Wen, H.-Y.; Liu, Y.-T.; Chang, Y.-C. Eur. J. Org. Chem. 2009,
1
14Z,17E,21-dien-3ꢀ-ol (8) standards by H and 13C NMR,
as well as GC-MS.3j The first novel new compound,
independently isolated from ERG7F699N and ERG7F699M
mutants, was characterized with NMR and confirmed as
malabarica-14E,17E,21-trien-3ꢀ-ol (4), a 6-6-5 tricyclic
product with chair-chair conformation and ∆14,17,21 double
bonds (see Supporting Information). This work describes the
first truncated intermediate of the oxidosqualene cyclases-
catalyzed cyclization/rearrangement cascade that alters the
substrate conformation from a ring-B boat conformation to
a ring-B chair conformation. The second novel compound
identified from ERG7F699M/N mutants was characterized as
17R-protosta-20,24-dien-3ꢀ-ol (5), a product with ∆20,24
double bonds and a C-17R hydrocarbon side chain config-
uration. Interestingly, this is the first reported product derived
from the protosteryl cation containing a C-17R-side chain
conformation, abrogating its further rearrangement reaction,
in ERG7 mutants. The third acetylated compound was
characterized as protosta-17(20),24-dien-3ꢀ-acetate. This
acetylated derivative was then hydrolyzed to yield its native
nonsaponifiable lipid, (17Z)-protosta-17(20),24-dien-3ꢀ-ol
(6). Recently, Lodeiro et al. also reported the characterization
of compound 3 and 6 from the oxidosqualene cyclases of
Aspergillus fumigates.5t
5731–5737
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Table 1 shows the product profiles produced from the
ERG7F699X site-saturated mutants. No product was observed
for the nonviable mutants, except for the ERG7F699N mutant,
indicating the abolished cyclization function of these mutated
ERG7 cyclases. The ERG7F699N mutant required exogenous
ergosterol to maintain its viability and produced 3, 4, 5, and 6
in the ratio of 55:5:24:16 without any compound 2. In the case
of the viable mutants, the ERG7F699L, ERG7F699I, and ERG7F699P
mutants produced 2 as their sole product, whereas the
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Org. Lett., Vol. 12, No. 3, 2010
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portion of this intermediate directly abstracts the C-15 proton,
with varying predisposition, and results in the production of 7
and 8. Alternatively, the cation Ib proceeds with a C-ring
expansion and is followed by D-ring annulations (II) to generate
the protosteryl C-20 cation with different stereochemical control
at the C-17 position (IIIa and IIIb). Notably, in the ERG7F699M
and ERG7F699N mutants, IIIa with a 17R- exocyclic hydrocarbon
side chain proceeds the deprotonated termination to produce 5.
Conversely, in most viable mutants, the protosteryl C-20 cation
is formed with a 17ꢀ- side chain conformation IIIb. Subse-
quently, a backbone rearrangement of H-17R to H-20R via a
1,2 hydride shift generates the protosteryl C-17 cation IV.
Elimination of a proton at C-13 or C-20 yielded 3 and 6,
respectively, as the major end products in most of the mutants.
Then, subsequent skeletal rearrangements of a hydride shift
(H13RfH17R), two methyl-group shifts (Me14ꢀfMe-13ꢀ and
Me-8RfMe-14R), and another hydride shift (H-9ꢀfH8ꢀ),
generate the lanosteryl C-8/C-9 cation (V), which undergoes
deprotonation at C-9 or C-8 to form 2, a normal biosynthetic
product.
Although it is difficult to rationalize the effect of a single
amino acid substitution on the production of diverse product
patterns, the illustration of the structure-function-mechanism
relationships through sequence alignment and homology
modeling might provide suitable insights for examining the
experimental results (Figure 1). First, multiple sequences
alignment results revealed that Phe699 of ERG7 is highly
conserved among all known triterpene cyclases. Nevertheless,
a single residue deletion was observed between Gly697 and
Val698 of ERG7, as compared with the corresponding region
of SHC (Figure 1a). Second, the homology structural model
of the ERG7 complexed with cation IV suggested that the
Phe699 of ERG7 is spatially positioned below the molecular
plane of the substrate with a distance of ∼4.8 Å between
the observed phenyl side chain and the C-17 position of IV
(Figure 1b).3h The π-electron rich phenyl group is optimal
for the stabilization of the electron-deficiency of IV, although
Table 1. Product Profiles of S. cereVisiae ERG7F699X Mutantsa
amino acids substitution
2
3
4
5
6
7
8
wtPhe
Leu
Ile
Asn
His
Thr
Met
Pro
100
100
100
-
-
-
-
55
70
-
-
-
5
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
24 16
13
-
-
1
17
-
<0.2 >99.8
1
100
46
-
10 17 18
-
-
-
-
-
a Neither cell viability nor product was characterized for the rest of the
ERG7F699X mutants.
ERG7F699H mutant produced 2, 3, and 6 in the relative ratio of
13:70:17. To our surprise, the ERG7F699M mutant generated
many different products including 2, 3, 4, 5, 6, 7, and 8. Notably,
for the viable ERG7F699T mutant, compound 3 accounted for
nearly one hundred percent (>99.8%) of its products, with
lanosterol (2) accounting for <0.2% as previously described.3h
On the basis of the product characterization, two alternative
cyclization/rearrangement pathways were proposed for the
ERG7F699X site-saturated mutants (Scheme 1). In the ERG7F699X
Scheme 1. Proposed Cyclization/Rearrangement Pathways of
Oxidosqualene within S. cereVisiae ERG7F699X Mutants
mutants, with the exception of the Asn and Met substitutions,
OS enters the enzyme active site cavity with a prefolded
C-B-C conformation. In the ERG7F699M/N mutants, although
most of the substrate prefolds with the C-B-C conformation,
some of the substrate forms an enforced C-C-C conformation
before the initial electrophilic epoxide activation. In one way,
the C-C-C substrate conformer is cyclized to a C-C 6-6-5
tricyclic Markonikov C-14 cation Ia, that is followed by direct
abstraction of proton from C-15 to yield 4 as the end product.
On the other hand, the C-B-C substrate conformer is cationic
cyclized to a C-B 6-6-5 tricycic Markonikov C-14 cation
(Ib) as the first stopping point. In the ERG7F699M mutant, a small
Figure 1. (A) Partial sequences alignment among HsOSC,
SceERG7, and AaSHC. (B) The superimposed homology models
of SceERG7 and SceERG7F699M complexed with protosteryl C-17
cation.
502
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Thorma et al. have proposed that the Phe699 side chain is
positioned to stabilize the anti-Markovnikov C-13 cation
during the C-ring formation.7a In addition, the His234 is
hydrogen bonded to Tyr510 and located spatially above the
molecular plane near the C-13 and C-20 positions of the
substrate.3d,f,7a Coupling with the Tyr99 positioned spatially
in the middle side wall proximal to the C-ring and the Tyr707
affecting the B-ring, enables these residues to form a
restricted girdle around the B/C/D ring region and exocyclic
side chain.3i Third, the structure comparison between ERG7
and SHC showed that the ꢀ-sheet domain of the ERG7
Gly697-Cys703 region is more loosely packed than the
corresponding region of SHC.6d The one amino acid deletion
between Gly697 and Val698 of ERG7 might promote the
position of Phe699 closer to the methyl group during the
B/C ring formation and also result in less space between
Phe699 and Tyr707, or between Phe699 and Tyr99, for the
orientation of the resulting methyl group. Consistent with
the observation is the isolation of a bicyclic intermediate from
the ERG7Y707X mutants, supporting the functional role of
Tyr707 in stabilizing both the bicyclic C-8 cation and the
final lanosteryl C-8/C-9 cation intermediates.3i Nevertheless,
genetic analysis and product characterization of both
ERG7F699M/Y707H and ERG7F699M/Y707Q double mutants showed
that both mutants cause yeast nonviability and no product
formation. In parallel, an Ala substitution of Phe601 from
SHC (corresponding to Phe699 of ERG7) also interrupted
the polycyclization cascade at either the tricyclic or tetra-
cyclic ring stage, resulting in the isolation of two premature
6/6/5 malabarica-14(27),17,21-triene derivatives with dif-
ferent stereochemically controlled exocyclic side-chain and
a 6/6/6/5-fused tetracyclic 17-isodammara-20(21),24-diene,
consistent with our results from the ERG7F699X mutants.9,10
Taken together, these results indicated that Phe699, in
conjunction with Tyr99, His234, and Tyr707, may play a
functional role in restricting the C-B-C conformation and/
or side chain rotation as well as in stabilizing the protosteryl
C-17 cation. The substitution of Phe699 with Asn, Met, His,
and Thr might cause inappropriate positioning of the side
chain and/or interrupt steric or electrostatic interaction within
the active site cavity, just allowing the unconstrained folding
of the substrate. Specifically, substitution of Phe699 to Asn
or Met might partially disrupt the transient dipole interactions
between the carbocationic intermediate and the hydroxyl
group of Tyr99 and/or of Tyr707, which in turn affects the
substrate folding and cyclization, resulting in the accumula-
tion of C-C-C or C-B-C conformor-induced tricyclic,
stereochemically inverted tetracyclic, and truncated rear-
ranged tetracyclic products. Or, mutant enzymes may have
more capacity to tolerate energetically unfavorable conform-
ers than wild-type due to the perturbance of their cyclization
energies prior to or during D-ring closure, resulting in the
isolation of intermediates arrested at tricyclic stage with
C-C-C conformation and a 17R epimer. Subsequent
misfolding of the exocyclic side chain might terminate the
subsequent rearrangement steps of the protosteryl C-20 cation
IIIa and result in the production of 5. However, other factors,
including steric or electrostatic effects from other residues
of the enzyme, which might be involved in the alternation
of the prefolded substrate conformation and subsequent
cyclization to diverse product profiles, could not be excluded.
In conclusion, our study demonstrate, for the first time,
that a single amino acid mutation at the 699 position of S.
cereVisiae ERG7 can change the course of the oxidosqualene
cyclization/rearrangement process on traversing the mecha-
nistic barriers between the prefolded C-B-C and C-C-C
substrate conformation, and between the 17R and 17ꢀ
stereochemistry of exocyclic hydrocarbon side chain. The
isolation of various tricyclic compounds with opposite B-ring
conformation demonstrates the possibility of traversing the
catalytic specificity between two distinct yet evolutionarily
related triterpene cyclases through molecular directed evolu-
tion or site-specific mutation of critical residues surrounding
the active site cavity. Finally, products with tailored specific-
ity or expanded diversity could only be simultaneously
obtained via the substitution of all proteinogenic amino acids
at some specific positions.
Acknowledgment. We are grateful to Dr. John H. Griffin
and Prof. Tahsin J. Chow for helpful advice. We thank the
MOE ATU Plan, the National Chiao Tung University as well
as the National Science Council of the Republic of China
for financial support of this research under Contract No.
NSC-97-2627-M-009-002, and NSC-97-2627-M-009-003.
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