The Botrytis cinerea type III polyketide synthase shows unprecedented high catalytic efficiency toward long chain acyl-CoAs

Article abstract of DOI:10.1039/c2mb25282a

BPKS from Botrytis cinerea is a novel type III polyketide synthase that accepts C₄-C₁₈ aliphatic acyl-CoAs and benzoyl-CoA as the starters to form pyrones, resorcylic acids and resorcinols through sequential condensation with malonyl-CoA. The catalytic efficiency (k_cat/K _m) of BPKS was 2.8 × 10⁵ s^-1 M ^-1 for palmitoyl-CoA, the highest ever reported. Substrate docking analyses addressed the unique features of BPKS such as its high activity and high specificity toward long chain acyl-CoAs.

Full text of DOI:10.1039/c2mb25282a

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COMMUNICATION
The Botrytis cinerea type III polyketide synthase shows unprecedented
high catalytic efficiency toward long chain acyl-CoAsw
a
a
a
a
b
Marimuthu Jeya, Tae-Su Kim, Manish Kumar Tiwari, Jinglin Li, Huimin Zhao* and
Jung-Kul Lee*a
Received 17th July 2012, Accepted 23rd August 2012
DOI: 10.1039/c2mb25282a
BPKS from Botrytis cinerea is a novel type III polyketide
synthase that accepts C –C18 aliphatic acyl-CoAs and benzoyl-
which shows the highest catalytic efficiency ever reported
4
toward long chain acyl-CoA esters.
CoA as the starters to form pyrones, resorcylic acids and
resorcinols through sequential condensation with malonyl-CoA.
Results
5
ꢁ1
ꢁ1
The catalytic efficiency (kcat/K
m
) of BPKS was 2.8 ꢀ 10 s
M
for palmitoyl-CoA, the highest ever reported. Substrate
docking analyses addressed the unique features of BPKS
such as its high activity and high specificity toward long chain
acyl-CoAs.
The amino acid alignment of BPKS with plant, bacterial and
fungal type III PKSs showed conservation of the Cys-His-Asn
catalytic triad in BPKS, evidence of BPKS being a type III
PKS (Fig. S1, ESIw). Alignment of BPKS with other type III
PKSs also revealed a carboxy-terminal extension of approxi-
mately 80 residues of BPKS. BPKS is longer than other type III
PKSs known to have C-terminal extensions: 1,3,6,8-tetra-
hydroxynapthalene synthase (THNS) from Streptomyces
Type III polyketide synthases (PKSs) generate the backbones of a
variety of plant secondary metabolites including chalcones, stil-
benes, phloroglucinols, resorcinols, benzophenones, biphenyls,
1
3
14
1,2
coelicolor and ORAS. BPKS cDNA was cloned without
C-terminal extension. The 1263 bp fragment of BPKS was
cloned and expressed in E. coli BL21-CodonPlus (DE3)-RIL
bibenzyls, chromones, acridones, pyrones, and curcuminoids.
The chalcone synthase (CHS) superfamily of type III PKS
enzymes are homodimers of 40–45 kDa proteins that directly
3–5
catalyze CoA-linked substrates using a single active site cavity.
with an N-terminal 6ꢀHis affinity tag. The purified His -tagged
6
BPKS protein gave a single protein band at B45 kDa on SDS-
The functional diversity of the CHS superfamily is attributable to
differences in their selection of starter substrates, the number
of polyketide chain extensions, and the mechanisms of their
cyclization reactions.
PAGE (Fig. S2, ESIw). The truncation of BPKS did not affect
the catalytic properties of the enzyme. The activity of the full
length enzyme and the truncated version was almost the same
6
7
(
data not shown), which suggests that the 80 amino acids at the
The genomes of Neurospora crassa and Aspergillus oryzae
8
C-terminal extension are not essential for the enzymatic activity
of BPKS. Similar findings were previously reported in other
predict the presence of type III PKSs in these filamentous fungi.
To date few fungal type III PKSs have been characterized,
examples being 2-oxoalkylresorcylic acid synthase (ORAS)
1
3,14
type III PKSs.
Various starter-CoAs were tested for their reactivity toward
BPKS. For C and C starter units, BPKS yielded a
9
–11
from N. crassa, CysA and CysB from Aspergillus oryzae
1
2
2
, C4,
C
6
8
and AnPKS from Aspergillus niger. To further examine the
catalytic mechanisms of fungal type III PKSs and to use these
enzymes for polyketide biosynthesis, additional fungal type III
PKSs need to be identified. This work reports the characteri-
zation of the gene product (BPKS) of XM_001555277.1
from Botrytis cinerea. This is the first report that describes
the catalytic properties of a type III PKS from B. cinerea,
single product. For longer starters (C10 to C14-CoA), two
products with similar UV absorption profiles were produced.
The HPLC profile of the product from hexanoyl-CoA (2b)
showed a single major peak at 280 nm (Fig. S3A, ESIw). Upon
LC/MS analysis, this product showed a molecular ion peak
ꢁ
(
M – H) at m/z 181.1, attributable to a cyclized triketide (3b)
synthesized from one molecule of hexanoyl-CoA and two
molecules of malonyl-CoA. Triketide (3d) (M = 265.0) and
tetraketide (4d) (M = 307.2) pyrones (Fig. S3B, ESIw) were
formed from lauroyl-CoA (2d) by BPKS. Other than tri- and
tetraketide pyrones, BPKS produced pentaketide resorcylic
acids and resorcinols from the C16–C18 starter-CoA substrates.
A hexaketide resorcylic acid was also produced from C18-CoA (2g).
Fig. 1 shows the HPLC profile of the products synthesized by
r
a
Department of Chemical Engineering, Konkuk University,
r
Seoul 143-701, Korea. E-mail: jkrhee@konkuk.ac.kr;
Fax: +82-2-458-3504
University of Illinois, Departments of Chemical and Biomolecular
Engineering, Chemistry and Bioengineering, Institute for Genomic
Biology, Center for Biophysics and Computational Biology,
b
600 South Mathews Avenue, Urbana, IL 61801, USA.
E-mail: zhao5@illinois.edu; Fax: +1 217-333-5052
BPKS from C -CoA. The HPLC profiles of other starters are
18
w Electronic supplementary information (ESI) available. See DOI:
1
0.1039/c2mb25282a
given in ESI.w Additionally, acetoacetyl-CoA and benzoyl-CoA
2
864 Mol. BioSyst., 2012, 8, 2864–2867
This journal is c The Royal Society of Chemistry 2012

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Fig. 1 HPLC analysis of the products synthesized by BPKS from
stearoyl-CoA. The inset figures show the MS/MS spectra and chemical
0
structure of the 5-(2 -oxononadecyl)-resorcinol (7g). MS/MS data of
other major products (3g, 4g, and 8g) are given in ESI.w
were tested with BPKS. Acyl-CoA with an aromatic side
chain, benzoyl-CoA (2h), could also be incorporated by
BPKS, generating a triketide (3h, M = 188) (Fig. S3C, ESIw).
r
Acetoacetyl-CoA (2i), whose structure mimics an intermediate
in the polyketide elongation pathway, was also used by BPKS,
yielding a triketide (3i, M = 126; Fig. S3D, ESIw). The exact
r
molecular weights of all identified products were confirmed
by HRMS (see ESIw). BPKS showed maximum activity with
palmitoyl-CoA (based on its initial rate of free CoASH
production) as a starter substrate. Table S1 (ESIw) lists the
relative activity of BPKS with various starter CoAs. BPKS
preferred long chain substrates for the synthesis of alkylresorcinols,
while the a-pyrones were the major products of reactions
primed with short and medium chain acyl-CoAs. The summary
of reactions catalyzed by BPKS is shown in Fig. 2.
Fig. 2 Summary of BPKS reactions with various acyl-CoA starter
substrates.
Table 1 Steady state kinetic parameters of BPKS protein with
different starter units. Results are means (n = 3) with S.E. values less
than 15%
ꢁ1
ꢁ1
ꢁ1
M )
Starter CoA
kcat (s
)
K
m
(mM)
k
cat/K
m
(s
Starter unit specificity was investigated through steady state
kinetic analyses of C –C acyl-CoAs (Table 1). Under optimal
3
Acetoacetyl-CoA
Benzoyl-CoA
Butyryl-CoA
Hexanoyl-CoA
Decanoyl-CoA
Myristoyl-CoA
Palmitoyl-CoA
Stearoyl-CoA
0.04 ꢂ 0.01
0.09 ꢂ 0.01
0.03 ꢂ 0.01
0.05 ꢂ 0.01
0.10 ꢂ 0.01
0.43 ꢂ 0.02
0.92 ꢂ 0.05
0.75 ꢂ 0.03
36.6 ꢂ 1.5
19.8 ꢂ 1.3
3.8 ꢂ 0.3
1.6 ꢂ 0.1
2.0 ꢂ 0.1
3.3 ꢂ 0.1
3.3 ꢂ 0.3
3.7 ꢂ 0.4
1.1 ꢀ 10
4.6 ꢀ 10
7.9 ꢀ 10
3.1 ꢀ 10
5.0 ꢀ 10
1.3 ꢀ 10
2.8 ꢀ 10
2.0 ꢀ 10
3
3
4
4
5
5
5
4
18
assay conditions (50 mM Tris-HCl, pH 8.0, and 30 1C), the highest
ꢁ
1
5
ꢁ1
ꢁ1
M ) were observed for
kcat (0.95 s ) and kcat/Km (2.8 ꢀ 10 s
palmitoyl-CoA. Km values of BPKS for hexanoyl-CoA and
palmitoyl-CoA starters were 1.6 mM and 3.3 mM, respectively.
K
m
values of various type III PKSs for acyl-CoA starters range
from 9.9 to 866.5 mM. Table S2 (ESIw) compares the catalytic
The specificity
9,15–17
properties of BPKS with those of other PKSs.
RppA and PKS18, which has been reported to accommodate
3–15
1
of BPKS toward long-chain aliphatic acyl-CoA (C14 to C18
)
the binding of long chain acyl-CoAs.
When the modeled
substrates is unprecedented in the fungal type III PKSs. ORAS
did not accept CoAs with aromatic side chains whereas BPKS
produced a triketide product with a catalytic efficiency of
BPKS structure was superimposed onto the structure of ORAS,
all the residues lining the active site cavity were well aligned; in
particular, the catalytic triads, Cys-His-Asn (located at posi-
tions 165, 317, and 350, respectively, in BPKS), overlapped
excellently. To investigate the molecular basis for the high
activity of BPKS toward long chain substrates, stearoyl-CoA
was docked into the substrate binding pocket of BPKS and
ORAS, then variations in the orientations of catalytic residues
between ligand bound BPKS and ORAS were examined.
Fig. S4 (ESIw) shows the superimposition of BPKS modeled
structure with bound stearoyl-CoA (C18 acyl-CoA) and ORAS
structure co-crystallized with eicosanoic acid (C20 fatty acid).
The acyl binding tunnel is lined by Phe265 (Phe252), Phe223
(Phe210), Cys133 (Cys120), Ser199 (Thr187) and Thr78 (Asn65)
residues. It validates that the docked stearoyl-CoA in the active
3
ꢁ1
ꢁ1
4
.5 ꢀ 10 s
M using benzoyl-CoA as a starter unit. Among
the fungal type III PKSs, next to AnPKS, BPKS showed the
highest activity with the aromatic starter unit.
Previous work has shown that the active site cavity of
type III PKSs acts as a size-based filter, determining starter
substrate specificity, the number of possible extensions and the
5
type of cyclization. To investigate the structural basis for the
observed substrate specificity, the homology model of BPKS
was built based on the only crystal structure showing high
sequence identity with BPKS: ORAS from N. crassa (PDB
code 3E1H; 65% identity). The modeled structure of BPKS
showed a similar acyl binding tunnel to that found in ORAS,
This journal is c The Royal Society of Chemistry 2012
Mol. BioSyst., 2012, 8, 2864–2867 2865

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been observed between the thioester carbonyl oxygen of
stearoyl-CoA and Asn338. The CoA part of stearoyl-CoA
˚
formed hydrogen bonds only with Thr310 (3.4 A) and Tyr54
˚
2.0 A) residues. Palmitoyl-CoA was also docked into the
(
˚
substrate binding tunnel of BPKS. A hydrogen bond (2.7 A)
was observed between the thioester carbonyl oxygen and
˚
nitrogen of Asn350. A distance of 2.9 A was observed between
the sulfur of the catalytic cysteine and the thioester carbonyl
group of palmitoyl-CoA. Several hydrogen bonding inter-
actions were also observed (Fig. S6, ESIw). Previous studies
on type III PKSs have shown that decreasing or increasing the
number of CoA interactions may alter the kinetics of the
association and dissociation of CoA thioesters and thus
5
influence the fate of the type III PKS reaction intermediates.
The docking analyses support the high activity of BPKS
toward palmitoyl-CoA and stearoyl-CoA.
Crystallographic and functional studies of CHS indicate
that a cysteine–histidine pair (Cys164-His303) forms part of
1
8
the catalytic machinery. It is reported that the active site
Cys164 nucleophile should be within the hydrogen bonding
distance of His303 so that His303 Ne can form a stable
imidazolium–thiolate ion pair with Cys164. In the crystal
1
9
structure of CHS, the Sg of Cys164 formed a hydrogen bond
˚
(
3.5 A) with the Ne of His303. We determined the distance
between Sg of Cys (165 in BPKS and 152 in ORAS) and Ne of
His (317 in BPKS and 305 in ORAS) in BPKS and ORAS
Fig. 3 Substrate docking of BPKS and ORAS in the substrate
binding tunnel. (A) Stearoyl-CoA docked in the substrate binding
tunnel of BPKS. (B) Stearoyl-CoA docked in the substrate binding
tunnel of ORAS. Hydrogen bonds between stearoyl-CoA and amino
acid residues are shown as green dotted lines. Stearoyl-CoA inter-
actions with catalytic residues are shown as solid lines. Amino acid
residues are shown in stick model colored grey while catalytic amino
acid residues are shown with carbon in green color. Stearoyl-CoA is
represented in ball and stick with carbon in blue color.
˚
˚
structures. Distances of 3.4 A and 3.8 A were observed in
BPKS and ORAS, respectively. This suggests that a more
stable imidazolium–thiolate ion pair can be seen in BPKS
(
Cys165-His317) when compared to ORAS.
Pyrone structures have been found to be inhibitors, such as
2
0
HIV-1 protease, metalloprotein, human leukocyte elastase,
2
and porcine pancreatic elastase, making them unique lead
1
structures for bioactive agents. Anti-cancer effects of alkyl-
2
resorcinols have been elucidated in previous reports.
2,23
BPKS,
site pocket of BPKS modeled structure has a similar confor-
1
mation to that of eicosanoic acid bound in ORAS.
as a type III PKS, exhibits the highest catalytic efficiency toward
aliphatic CoAs than any other PKSs and thus is a potential
biocatalyst for the synthesis of alkyl-pyrones and resorcinols.
The product profiles can be controlled based on the length of
the starter units.
4
When stearoyl-CoA was docked into the substrate binding
site of BPKS (Fig. 3A), stearoyl-CoA was bound through
˚
hydrogen bonds (green line) with Gly319 (2.1 A), Ala321
˚ ˚ ˚
3.4 A), and Thr322 (2.8 A). A hydrogen bond (3.2 A) has
(
been observed between the thioester carbonyl oxygen and
˚
nitrogen of Asn350. While a distance of 2.9 A was observed
Conclusions
between the sulfur of the catalytic cysteine and the thioester
carbonyl group of stearoyl-CoA which is essential to start the
nucleophilic attack. Other intramolecular interactions were
also seen. Fig. 3A shows that the residues Thr322, Ala321
and Gly319 contribute hydrogen bonds to CoA binding. All
these interactions position the CoA molecule at the entrance to
the BPKS active site and orient the C18 stearoyl moiety at the
end of the CoA tunnel that opens into a cavity that defines the
acyl binding tunnel. The other interactions observed between
the bound stearoyl-CoA and the residues in the acyl-CoA
binding tunnel are shown in Fig. S5 (ESIw). In ORAS, how-
ever, there was no hydrogen bond between oxygen of the
phosphate and the Ala residue (Fig. 3B). Moreover, the
distance of the thioester carbonyl group of stearoyl-CoA from
In this study a putative type III PKS from B. cinerea was
characterized. It synthesized fatty acyl-primed tri, tetraketide
pyrones, resorcylic acid and resorcinol compounds from various
starter units. Although BPKS displayed broad substrate
specificity, it exhibited highest activity toward long chain
length starter units. Homology modeling studies of BPKS
with stearoyl-CoA shed light on the high activity of BPKS
toward stearoyl-CoA. Crystal structure analyses combined
with mutational studies are now in progress to probe the
structural basis for the catalytic properties of BPKS.
Acknowledgements
This work was supported by the National Research Founda-
tion of Korea (NRF) grant funded by the Korea government
(MEST) (220-2009-1-D00033).
˚
the catalytic Cys152 was longer (3.9 A) compared to the
˚
distance observed in BPKS. A hydrogen bond (3.8 A) has
2
866 Mol. BioSyst., 2012, 8, 2864–2867
This journal is c The Royal Society of Chemistry 2012

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