Functional analysis of phenylalanine 365 in hopene synthase, a conserved amino acid in the families of squalene and oxidosqualene cyclases

Article abstract of DOI:10.1039/a905559b

Two bicyclic products were accumulated by the mutant F365A, showing the amino acid residue is located close to the transient C-8 carbocation intermediate in the active site cavity; the mutants of F365Y and F365W significantly accelerated the cyclization reaction at low temperatures.

Full text of DOI:10.1039/a905559b

Functional analysis of phenylalanine 365 in hopene synthase, a conserved
amino acid in the families of squalene and oxidosqualene cyclases†
Tsutomu Hoshino* and Tsutomu Sato
Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and
Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan. E-mail: hoshitsu@agr.niigata-u.ac.jp
Received (in Cambridge, UK) 9th July 1999, Accepted 3rd September 1999
Two bicyclic products were accumulated by the mutant
F365A, showing the amino acid residue is located close to the
transient C-8 carbocation intermediate in the active site
cavity; the mutants of F365Y and F365W significantly
accelerated the cyclization reaction at low temperatures.
bacillus acidocaldarius has been reported and a reaction
mechanism proposed.5 However, site-directed mutagenesis
experiments are required to verify the catalytic functions of the
residues of the presumed active center. To date, only a few
essential components of these active sites have been demon-
strated by point mutation experiments.4b,6 The polycyclization
cascade has been proposed to proceed by the stabilization of
discrete carbocation intermediates via cation–p interaction with
aromatic amino acid residues of the cyclases,7 but no kinetic
evidence has been reported. In a series of point mutation
experiments conducted by us, Phe365, which is conserved
among all the known prokaryotic SHCs and eukaryotic
cyclases,2,5,6c has been identified as having a stabilizing
function for the transient C-8 carbocation intermediate. We
show here kinetic data of the mutated SHCs of F365Y and
F365W.
Cell-free homogenates of the E. coli clone encoding the
mutated F365A SHC, prepared from 8 L culture, were
incubated with 1 (105 mg) at pH 6.0 and 55 °C for 16 h. The GC
analysis showed the presence of two major products (4 and 5),
a little of 2 and recovered 1 in the reaction mixture. Separation
on a SiO2 column using hexane as an eluent afforded 4 (27.8
mg), 5 (25.1 mg), 2 (3.0 mg) and 1 (32.4 mg). Analyses of the
2D NMR spectra revealed that the structures of 4 and 5 had a
6/6-fused bicyclic skeleton.8 Formation of 4 and 5 suggested
that the sequential cyclization reactions are quenched at the
bicyclic stage of the putative intermediate 3. A series of
1,2-shifts of hydrides and a methyl group in an antiparallel
fashion could trigger the deprotonation at C-6 to produce 4,
while a deprotonation at C-7 could give rise to 5 (Scheme 1).
These findings indicate that the F365 residue is critical to the
completion of the polycyclization and that the p-electron of this
side chain may stabilize the transient C-8 carbocation. There
were no detectable amounts of mono- or tri-cyclic products,
further suggesting that the F365 residue is close to the C-8
cation in the enzyme cavity, but not near the mono- and tri-
cyclic intermediary carbocations, which is consistent with the
recent suggestion inferred from X-ray analysis.5
The cyclization of squalene 1 into the pentacyclic triterpene
hopene 2, mediated by the prokaryotic squalene-hopene cyclase
(SHC), is one of the most sophisticated biochemical reactions
(Scheme 1).1 Oxidosqualene also undergoes the polyolefin
cyclization by eukaryotic cyclases, in a similar manner to 1.1
Recent investigations on the molecular biology of the two
cyclases2 and on the substrate analogues1,3 have encouraged
mechanistic studies on the polycyclization reactions. The
acyclic molecule 1 is believed to be folded into an all pre-chair
conformation inside the enzyme cavity,1 but the involvement of
a ring expansion process has recently been disclosed with
respect to the D-ring formation.4a,b Such a ring expansion
reaction also occurs in C-ring formation by lanosterol syntha-
se.4c,d An X-ray crystal structure of the SHC from Alicyclo-
To validate the proposed role of the cation–p interaction, the
mutants F365Y and F365W were constructed based on the idea
that the higher electron density of the p–electrons, the faster
cyclization reaction. Fig. 1 depicts the specific activities of
these mutants against incubation temperatures. The mutant
F365Y completed the polycyclization to the final product 2
without any intermediate being observed, differing from
F365A, and exhibited a remarkable increase in the reaction rate
at low temperatures (10–50 °C); the F365Y produced 2 even at
a temperature as low as 10 °C, a temperature at which the wild-
type SHC has no significant activity. The activation energy
(Eact), estimated from the Arrhenius plots,9 was greatly reduced;
e.g. 37.0 kJ mol21 for the F365Y compared with 50.1 kJ mol21
for the wild-type. The higher electron density of the tyrosine
residue apparently gives rise to a faster cyclization reaction. The
Km values of the wild-type and F365Y were determined from
the Lineweaver–Burk plots to be 16.9 ± 0.6 (30–60 °C) and 502
± 12 mM (10–50 °C), respectively, suggesting that the substrate
affinity for F365Y was greatly decreased. An unusual profile
was found between activities and temperatures (Fig. 1). Three
Scheme 1
† Spectroscopic data for 4 and 5, Arrhenius plots for 1 and 6 and CD spectra
for the relevant SHCs are available from the RSC web site, see
http://www.rsc.org/suppdata/cc/1999/2005/
Chem. Commun., 1999, 2005–2006
This journal is © The Royal Society of Chemistry 1999
2005

The previously unknown 4 and the known products 5 (g-
polypodatetraene),13 which have a 6/6-fused bicyclic skeleton,
were produced by the site-directed mutant of F365A due to the
lack of p-electrons, suggesting that the major function of
Phe365 would be assigned for stabilization of the C-8
carbocation intermediate possibly via a cation–p interaction.
Notes and references
1 I. Abe, M. Rohmer and G. D. Prestwich, Chem. Rev., 1993, 93, 2189.
2 E. J. Corey, H. Cheng, C. H. Baker, S. P. T. Matsuda, D. Li and X. Song,
J. Am. Chem. Soc., 1997, 119, 1289.
3 (a) T. Hoshino and T. Kondo, Chem. Commun., 1999, 731; (b) B.
Robustell, I. Abe and G. D. Prestwich, Tetrahedron Lett., 1998, 39,
957.
4 (a) C. Pale-Grosdemange, C. Feil, M. Rohmer and K. Poralla, Angew.
Chem., Int. Ed., 1998, 37, 2237; (b) T. Sato, T. Abe and T. Hoshino,
Chem. Commun., 1998, 2617; (c) E. J. Corey and H. Cheng,
Tetrahedron Lett., 1996, 37, 2709; (d) T. Hoshino and Y. Sakai, Chem.
Commun., 1998, 1591.
5 K. U. Wendt, K. Poralla and G. E. Schulz, Science, 1997, 277, 1811;
K. U. Wendt, A. Lenhart and G. E. Schulz, J. Mol. Biol. 1999, 286,
175.
Fig. 1 Specific activities for the formations of 2 (closed symbols) and 7
(open symbols) against incubation temperatures are given for the wild-type
SHC (-, 8), the mutants of F365Y(5, 2) and F365W (:, Ω ). One mg of
1 or 6 was incubated with 5 mg of the homogeneously purified SHCs for 60
min at pH 6.0.
6 (a) C. Feil, R. Sussmuth, G. Jung and K. Poralla, Eur. J. Biochem., 1996,
242, 51; (b) T. Sato,Y. Kanai and T. Hoshino, Biosci. Biotechnol.
Biochem., 1998, 62, 407; (c) T. Sato and T. Hoshino, Biosci. Biotechnol.
Biochem., 1999, 63, 1171.
distinctive phases were observed: at 10–25°C, the activities
were increased; at 30–50 °C, a steady state was reached; at
55–70 °C, the activities decreased due to irreversible thermal
denaturation.10 The enzyme activity of the F365W was
negligible (Fig. 1) despite the higher electron density of the
indole ring, which may result from the significantly decreased
binding of 1 to the catalytic site. The CD spectra of the three
mutants were superimposable on that of the wild-type. How-
ever, significant local change may be indeed likely and this may
have no visible effect on the overall CD spectra.
(3S)-2,3-Oxidosqualene 6 also undergoes the cyclization by
SHC to form 3b-hydroxyhopene 7.3b,6c,11 Compound 6 was
incubated as another enzymic test to obtain further evidence for
the cation–p interaction. F365Y had a remarkably enhanced
activity, 5-fold higher than the wild-type, and showed a bell-
shaped curve (Fig. 1).10 As expected, F365W also had higher
activity than the wild-type at temperatures below 50 °C, but
exhibited an unusual profile analogous to that of 1 with the
F365Y.10 No intermediate products were detected in the
reaction mixtures of 6 by either F365Y and F365W, whereas the
3-hydroxy derivatives corresponding to bicyclic intermediates
were accumulated by the F365A without completion of the
polycyclization, further supporting the idea that the residue at
365 must be an aromatic amino acid. The values of Eact9 with 6
were 53.6, 49.9 and 42.8 kJ mol21, respectively, for the wild-
type, the F365Y and the F365W mutants, which is in good
agreement with the cation–p interaction concept. The enhanced
Km values for these mutants were also observed with 6; the Km
of the F365W was largest.11
7 D. A. Dougherty, Science, 1996, 271, 163.
8 Analyses of NMR data (H-H COSY 45, HOHAHA, NOESY, DEPT,
HMQC and HMBC) unequivocally supported the structures proposed
for 4 and 5.
9 The temperatures for the Arrhenius plots for 1 were in the range of
30–60 and 10–25 °C for the wild-type and for the F365Y, respectively,
while in the case of 6 they were 40–60, 30–50 and 20–30 °C,
respectively, for the wild-type, the F365Y and the F365W.
10 Our working hypothesis for the interpretation of this unusual behaviour
is as follows. Reversible denaturation12b may gradually occur from 30 to
50 °C, and the denaturation may be attributable to the more enhanced
susceptibility of the mutants to the exothermic high energy, which is
released by the cyclization reaction,5 because the geometrical change
occurred at the active site region (significantly increased Km). As for the
F365Y, temperature dependency for the reaction is different between 1
and 6. This may be closely related to the increased degree of Km, leading
to the unusual profile for 1 (Km = 502 mM, more sensitive to higher
temperatures), but to a bell-shape for 6 (Km = 182 mM, less sensitive).
If the denaturation could not occur, the kcat of 1 may have been
significantly increased at 30–50 °C. This idea may also be true for the
case of 6 by the F365W (Km = 357 mM).11 The denaturation process
was confirmed to be reversible; the F365Y was exposed at 40 °C for 60
min and then incubated with 1 at 20 °C, but showed the same activity as
that without such treatment. To validate our hypothesis, further evidence
is required.
11 Kinetic data for the reactions of 6, measured at 40 °C and for 60 min, are
as follows: kcat 1.4, 12.2 and 17.2 min21, and Km 0.72, 181.8 and 357.2
mM, respectively, for the wild-type, F365Y and F365W. The more bulky
the substituents, the larger the values of Km. The kcats and Kms of the
wild-type for the reactions of 1 and 6 at 60 °C are as follows: 288.5
min21 and 16.7 mM for 1; 23.6 min21 and 0.84 mM for 6.6c
12 (a) P. W. Hochachka and G. N. Somero, in Biochemical Adaptation,
Princeton University Press, 1984; (b) K. Hiromi, Y. Takasaki and S.
Ono, Bull. Chem. Soc. Jpn., 1963, 36, 563; W. B. Neely, J. Am. Chem.
Soc., 1959, 81, 4416.
The faster cyclization reactions at lower temperatures with
the Tyr and the Trp mutants suggest that the cation–p
interaction is likely to occur for the squalene cyclization
cascade. The bulky aromatic substituents make the active site
region less compact. A somewhat loosely packed protein
structure may be more flexible at low temperatures leading to
high catalytic activity, as is found for psychrophilic enzymes.12a
More detailed studies are required to gain greater insight into
the kinetics of these mutants.10
13 K. Shiojima, Y. Arai, K. Masuda, T. Kamada and H. Ageta,
Tetrahedron Lett., 1983, 24, 5733.
Communication 9/05559B
2006
Chem. Commun., 1999, 2005–2006