Protostadienol synthase from Aspergillus fumigatus: Functional conversion into lanosterol synthase

Article abstract of DOI:10.1016/j.bbrc.2009.11.160

Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3β-ol which is the precursor of the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues ⁷⁰²APPGGMR⁷⁰⁸ were replaced with ⁷⁰²NKSCAIS⁷⁰⁸, as in human OSLC. As a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease in the V_max/K_M value, whereas the binding affinity for the substrate did not change significantly. Homology modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-π interactions is important for the product outcome between protostadienol and lanosterol.

Full text of DOI:10.1016/j.bbrc.2009.11.160

Biochemical and Biophysical Research Communications 391 (2010) 899–902
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Protostadienol synthase from Aspergillus fumigatus: Functional conversion into
lanosterol synthase
*
Miki Kimura, Tetsuo Kushiro, Masaaki Shibuya, Yutaka Ebizuka, Ikuro Abe
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 November 2009
Available online 29 November 2009
Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cycliza-
tion of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3b-ol which is the precursor of the steroidal
antibiotic helvolic acid. To shed light on the structure–function relationship between OSPC and oxido-
squalene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues
702APPGGMR708 were replaced with 702NKSCAIS708, as in human OSLC. As a result, the mutant no longer
produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol.
This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease
in the Vmax/KM value, whereas the binding affinity for the substrate did not change significantly. Homol-
ogy modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701
Keywords:
Oxidosqualene cyclase
Lanosterol synthase
Protostadienol synthase
Steroidal antibiotics
Helvolic acid
through cation-
p
interactions is important for the product outcome between protostadienol and
lanosterol.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
from H-17a or H-13a to form a double-bond between C-17/C-20
or C-13/C-17, respectively.
Oxidosqualene cyclases (OSCs) are pivotal enzymes in the bio-
synthesis of sterols and triterpenes [1–4]. Oxidosqualene:lanosterol
cyclase (OSLC) catalyzes the cyclization of (3S)-2,3-oxidosqualene
into lanosterol, which is the key step in the biosynthesis of sterols
in animals and fungi (Fig. 1). The formation of lanosterol is initiated
by the oxirane ring opening of oxidosqualene folded in the chair–
boat–chair conformation; the sequential ring forming reaction first
produces the tetracyclic protosteryl C-20 cation, which then under-
To clarify the structure–function relationship between OSLC
and OSPC, and to understand the intimate structural details that
govern the stabilization of the C-20 protosteryl cation and the
backbone rearrangement reactions, we constructed a series of A.
fumigatus OSPC mutants and investigated the effects of the muta-
genesis on the enzyme activity. Here we report the functional con-
version of A. fumigatus OSPC into OSLC by replacing the C-terminal
residues 702APPGGMR708, located just downstream of the active-
site Phe701, with 702NKSCAIS708, as in human OSLC (Fig. 2). The
active-site Phe701 is considered to be important for stabilization
of the positive charges at the C-20 protosteryl cation through cat-
goes backbone rearrangement (H-17a ? 20, H-13a ? 17a, CH3-
14b ? 13b, CH3-8 ? 14 ) followed by removal of H-9 to generate
a
a
the D8 double bond of lanosterol. On the other hand, oxidosqua-
lene:protostadienol cyclase (OSPC) (AfuOSC3 or Afu4g14770) from
the fungus Aspergillus fumigatus is a recently reported novel OSC
which is involved in the biosynthesis of the fusidane-type steroidal
antibiotic helvolic acid [5,6]. OSPC is an Mr 83,086 membrane bound
protein with 735 amino acids, sharing 40% amino acid identity with
human OSLC. A recombinant OSPC heterologously expressed in an
OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae cat-
alyzed the cyclization of (3S)-2,3-oxidosqualene into a 3:1 mixture
of protosta-17(20)Z,24-dien-3b-ol and (20R)-protosta-13(17),24-
dien-3b-ol (Fig. 1) [5]. In this case, the cyclization of (3S)-2,3-oxido-
squalene proceeds without the backbone rearrangement, and the
C-20 protosteryl cation immediately undergoes proton elimination
ion-p interactions, which would be crucial for the formation of
lanosterol via the backbone rearrangement reaction [7]. The results
suggested that the swapping of the residues neighboring the
active-site Phe701 caused changes in the position of Phe701, there-
by affecting the cation-p interactions and the stabilization of the C-
20 cation, which led to the production of lanosterol by the OSPC
mutant.
Materials and methods
PCR and sequence analysis. The oligo DNAs were synthesized by
Nihon Bioservice (Saitama, Japan) and Invitrogen. PCR was per-
formed with a PTC-200 Peltier Thermal Cycler (MJ Research).
Sequencing was accomplished with an ABI PRISM 3100 Genetic
Analyzer (Applied Biosystem).
* Corresponding author. Fax: +81 3 5841 4744.
E-mail address: abei@mol.f.u-tokyo.ac.jp (I. Abe).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.11.160

900
M. Kimura et al. / Biochemical and Biophysical Research Communications 391 (2010) 899–902
H
H
20H
H
H
H
OSLC
17
13
9
cholesterol
ergosterol
8
H
H
HO
HO
HO
O
H
H
H
lanosterol
protosteryl cation
(3S)-2,3-oxidosqualene
H
OSPC
H
11
H
H
20
COOH
OAc
9
H
O
H
17
13
17
1:3
HO
H
H
H
H
parkeol
HO
H
O
HO
H
H
OAc
protosta-17(20)Z,24-dien-3β-ol
(20R)-protosta-13(17),24-dien-3β-ol
helvolic acid
Fig. 1. Proposed mechanism of cyclization of (3S)-2,3-oxidosqualene into lanosterol by OSLC and protosta-17(20)Z,24-dien-3b-ol and (20R)-protosta-13(17),24-dien-3b-ol by
OSPC.
Site-directed mutagenesis. The A. fumigatus OSPC mutant in
which the residues 702APPGGMR708 were replaced with 702NKS-
CAIS708, was constructed by a PCR method. Thus, the first PCR
AGTGTTTAATCCTCCTGGGGGGATGCGGTATCCAAATTAC-30; AfuOSPC-
P703 K/P704S, 50-CCGCTGGAAGGAGTGTTTGCTAAGTCTGGGGGGA
TGCGGTATCCAAATTAC-30; AfuOSPC-G705C, 50-CCGCTGGAAGGA
GTGTTTGCTCCTCCTTGCGGGATGCGGTATCCAAATTAC-30; AfuOSPC-
M707I, 50-CCGCTGGAAGGAGTGTTTGCTCCTCCTGGGGGGATCCGG
TATCCAAATTAC-30.
Enzyme expression. After confirmation of the sequence, the plas-
mid was transformed into the OSLC-deficient yeast mutant strain
GIL77 (erg7, ura3-167, hem3-6, gal2) using Frozen-EZ Transforma-
tion II kit (Zymo Research), and the cells were plated onto synthetic
complete medium without uracil (SC-U), containing ergosterol
was performed with
(AfuOSPC-C-Bgl: 50-GTACCAAGATCTTTATATAGCTAGACATTCATTT
CCATGG-30) and 1
L (20 pmol) of a mutation primer (AfuOSPC-
NKSCAIS-S: 50-CCGCTGGAAGGAGTGTTTAATAAGTCTTGCGCGATC
AGCTATCCAAATTAC-30) with 1
L of plasmid DNA containing the
1 lL (20 pmol) of a C-terminal primer
l
l
full length cDNA encoding A. fumigatus OSPC [5] as the template.
Phusion DNA polymerase (Finnzymes) was used with dNTP
(0.2 mM) in a final volume of 50 lL, according to the manufac-
turer’s protocol. The reaction proceeded for 30 cycles with the fol-
lowing program: 98 °C, 10 s, 60 °C, 24 s, 72 °C, 30 s, and final
extension at 72 °C, 10 min. The resulting 120 bp fragment was sep-
arated by agarose gel (2%) electrophoresis and purified using a
MonoFas DNA purification kit (GL Sciences). The second PCR was
(20 lg/mL), hemin chloride (13 lg/mL), and Tween 80 (5 mg/mL).
The cells harboring the plasmid were incubated at 30 °C for 2 days
for selection of the desired transformants. The culture of the trans-
formed yeast, the protein expression, and the isolation, purification
and GC-MS analysis of the products were performed as described
previously [5].
Enzyme preparation. A 2 L culture of GIL77 harboring each gene
was induced with galactose for 1 day and harvested. The collected
cells were suspended in 20 mL of 0.1 M KPB (pH 7.4) and the cells
were disrupted with a French Press (25,000 psi). After centrifuga-
tion (10,000g) to remove the cell debris, ultracentrifugation
(100,000g) was performed for 1 h to collect the microsome fraction
which was then suspended in 2 mL of 0.1 M KPB (pH 7.4) with 0.2%
Triton X-100. Enzyme solubilization was accomplished by vor-
texing for 1 h and then ultracentrifugation (100,000g) for 1 h. The
resulting supernatant was then passed through a hydroxylapatite
column (1 mL) equilibrated with 5 mM KPB (pH 7.4) and the flow
through fraction was collected and used for the enzyme reaction.
performed with 10
and 1
L (20 pmol) of the N-terminal primer (AfuOSPC-N-Spe, 50-
CTTGCCTACTAGTATGGCGACAGACAGCAGCATG-30) with
of
lL of this fragment as an anti-sense primer,
l
1 lL
plasmid DNA containing the full length cDNA encoding A. fumiga-
tus OSPC as the template. The reaction was performed for 30 cycles
with the following program: 98 °C, 10 s, 60 °C, 24 s, 72 °C, 30 s, and
final extension at 72 °C, 5 min. The resulting 2.3 kb band, corre-
sponding to the full length cDNA fragment was digested with SpeI
and BglII and ligated into the yeast expression plasmid pESC-URA
(Stratagene), which was previously digested with the same restric-
tion enzymes.
For the construction of point mutants of A. fumigatus OSPC, the
following primers were used. AfuOSPC-A702N, 50-CCGCTGGAAGG
Fig. 2. Comparison of the amino acid sequences of the C-terminal regions of the A. fumigatus OSPC and the OSLCs from a mammal, yeast, and fungi: Human, Homo sapiens;
Saccharomyces, Saccharomyces cerevisiae; Candida, Candida albicans; Cephalosporium, Cephalosporium caerulens; Afu, Aspergillus fumigatus. The active-site Phe701 (numbering
in A. fumigatus OSPC) is marked with #. The residues 702APPGGMR708 are marked with ꢀ.

M. Kimura et al. / Biochemical and Biophysical Research Communications 391 (2010) 899–902
901
Enzyme assay. For the steady-state kinetic analysis, 100
the solubilized enzyme solution was incubated with [14C]-(3S)-
2,3-oxidosqualene (10,000 dpm/5 L EGME solution) at various
l
L of
guiding the stereochemical course of the enzyme reaction [7]. In-
deed, the F696T mutant of S. cerevisiae OSLC (ERG7) (numbering
in human OSLC) was recently shown to produce a truncated rear-
rangement product, protosta-13(17),24-dien-3b-ol, which was
formed by hydride shift from C-17 to C-20 followed by proton
l
concentrations for 1 h at 30 °C. The reaction was quenched by
the addition of 20% KOH/EtOH, extracted with hexane and spotted
onto a TLC plate (Merck #11798), which was developed with ben-
zene:acetone = 19:1. Radioactivities were quantified by an imaging
plate (Fuji Film) and analyzed by a BAS-1500 system (Fuji Film).
Lineweaver–Burk plots of the data were utilized to derive the
apparent KM and Vmax values (average of triplicates standard
deviation).
elimination of H-13a [8,9]. We therefore surmised that the resi-
dues located around Phe701 might be participating in the fate of
the C-20 cation, and the difference between OSLC around this re-
gion might be responsible for the different product outcomes be-
tween the two enzymes. Thus, we first constructed a mutant of
A. fumigatus OSPC in which the residues 702APPGGMR708 were re-
placed with 702NKSCAIS708, as in human OSLC, and investigated
the effects of the mutagenesis on the enzyme activity.
As in the case of the wild-type A. fumigatus OSPC, the gene
encoding the mutant enzyme was cloned into the yeast expression
vector pESC(Ura) and heterologously expressed in the OSLC-defi-
cient sterol auxotrophic yeast mutant strain GIL77 ( erg7, ura3-
167, hem3-6, gal2) under the control of the GAL10 promoter [5].
After induction, the transformed cells were harvested and the re-
combinant membrane-bound enzyme was solubilized with Triton
X-100 and partially purified by hydroxylapatite column chroma-
tography as described previously [10].
GC-MS analysis. The 4,4-dimethyl sterol fractions were applied
to a GC-MS system (Shimadzu, GCMS-QP2010) equipped with a
Restec Rtx-5MS glass capillary column (30 m in length, 0.25 mm
in diameter, 0.25 lm film thickness) and using He as a carrier
gas (45 cm/min) with the following program: maintained at
240 °C for 2 min, followed by temperature increase at the rate of
10 °C/min up to 330 °C. The temperature of the ionization chamber
was 250 °C, with electron impact ionization at 70 eV.
Results and discussion
An in vitro enzyme assay revealed that the A. fumigatus OSPC mu-
tant in which the residues 702APPGGMR708 were replaced with the
conserved 702NKSCAIS708 sequence no longer produced protostadie-
nols, but instead efficiently produced a 1:1 mixture of lanosterol and
parkeol (Fig. 3). Indeed, the mutant complemented the erg7 defi-
ciency of the yeast GIL77 strain, since the transformed cells could
grow on media lacking ergosterol, in sharp contrast to the wild-type
OSPC. This is the first demonstration of the functional conversion of
OSPC into OSLC and the formation of lanosterol by the OSPC mutant.
Here the proposed mechanism of (3S)-2,3-oxidosqualene cycliza-
tion to parkeol is essentially the same as that for lanosterol; the only
difference is the final proton elimination from H-11 to produce the
double-bond between C-9/C-11 of parkeol, whereas proton elimina-
A comparison of the primary sequences of the A. fumigatus OSPC
and OSLC enzymes from a mammal, yeast and fungi revealed that
the moderately conserved C-terminal residues of human OSLC,
697NKSCAIS703, located just downstream of the active-site Phe696
(numbering in human OSLC), are characteristically replaced with
702APPGGMR708 in A. fumigatus OSPC (Fig. 2) [5]. In the X-ray
crystal structure of human OSLC, the active-site Phe696 (which
corresponds to Phe701 in A. fumigatus OSPC) is located near the
C-13/C-20 of the bound lanosterol. This Phe residue is considered
to be important for stabilization of the positive charges at the
C-13 anti-Markovnikov tricyclic cation and also at the C-20 tetra-
cyclic protosteryl cation through cation-p interactions, thereby
(x1,000,000)
TIC
6.0
protosta-17(20)Z,24-
dien-3 -ol
(20R )-protosta-
13(17),24-dien-3 -ol
5.0
4.0
3.0
2.0
1.0
9.00
9.25
9.50
9.75
10.00
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
(x10,000,000)
TIC
2.0
1.5
1.0
0.5
lanosterol
parkeol
9.00
9.25
9.50
9.75
10.00
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
Fig. 3. GC-MS trace of the enzyme reaction products of (A) the wild-type A. fumigatus OSPC and (B) the OSPC mutant in which the residues 702APPGGMR708 were replaced
with the conserved 702NKSCAIS708
.

902
M. Kimura et al. / Biochemical and Biophysical Research Communications 391 (2010) 899–902
cationic center of the protosteryl cation as compared with that in
human OSLC (Fig. 4). This increased distance would reduce both
the cation- interactions and the stabilization of the C-20 cation,
p
and hence would interrupt the backbone rearrangement, resulting
in termination of the enzyme reaction by proton elimination of H-
17a or H-13a to produce a 3:1 mixture of protosta-17(20)Z,24-
dien-3b-ol and (20R)-protosta-13(17),24-dien-3b-ol (Fig. 1). Alter-
natively, an activated water molecule might be located in a cavity
formed between the C-20 cation and Phe701, and abstract a proton
from C-17 or C-13 to terminate the reaction at the protosteryl cat-
ion stage. The relocation of the Phe701 residue in the mutant might
have prevented the water molecule from entering this cavity, thus
precluding the early termination of the reaction to produce lanos-
terol and parkeol.
In conclusion, this is the first report of the functional conversion
of OSPC to OSLC by changing the conserved C-terminal residues
702APPGGMR708 into 702NKSCAIS708. The results suggested that sta-
bilization of the C-20 protosteryl cation by the active-site Phe701
through cation-p interactions is important for the product out-
come between protostadienol and lanosterol. To further clarify
the structural details of the enzyme reactions, crystallization trials
of both the wild-type and mutant A. fumigatus OSPCs are now in
progress in our laboratories.
Acknowledgments
We thank Professor Tung-Kung Wu (National Chiao Tung Uni-
versity, Republic of China) for the gift of the authentic samples of
parkeol and protosta-13(17),24-dien-3b-ol. This work was sup-
ported by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology, Japan, and
by Grant-in-Aids from The Yamada Science Foundation, and the
Astellas Foundation for Research on Metabolic Disorders, Japan.
Fig. 4. (A) A homology model of A. fumigatus OSPC complexed with lanosterol. (B)
The X-ray crystal structure of human OSLC complexed with lanosterol. Only the
active-site Phe701 and the residues 702APPGGMR708 (numbering in A. fumigatus
OSPC) are indicated. Lanosterol is shown in magenta and the position of the C-20
indicated.
tion from H-9 generates the double-bond between C-8/C-9 of
lanosterol (Fig. 1). The steady-state kinetic analysis revealed that
the mutant had an apparent KM = 16.5 lM, Vmax = 0.32 l ,
M minꢁ1
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