Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Article abstract of DOI:10.1002/anie.201709636

While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β-keto group and dehydration of a β-hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11–Iga12), Iga13 and Iga16 with malonyl and hexanoyl-CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C₈, C₁₀, C₁₂, and C₁₄), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto-reduction and dehydration with strict stereo-specificity. We propose “highly reducing type II PKS subfamily” for the polyene-producing type II PKSs.

Full text of DOI:10.1002/anie.201709636

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Accepted Article
Title: Reconstitution of a Type II Polyketide Synthase Catalyzing
Polyene Formation
Authors: Danyao Du, Yohei Katsuyama, Kazuo Shin-ya, and Yasuo
Ohnishi
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Angewandte Chemie International Edition
10.1002/anie.201709636
COMMUNICATION
Reconstitution of a Type II Polyketide Synthase Catalyzing Poly-
ene Formation
[
a]
[a],
[b]
[a],
Danyao Du , Yohei Katsuyama *, Kazuo Shin-ya , Yasuo Ohnishi *
Abstract: While type II polyketide synthases (PKSs) are known for
producing aromatic compounds, a phylogenetically new subfamily
of type II PKSs have been recently proposed to synthesize polyene
structures. Here we report in vitro analysis of such a type II PKS,
IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and
dehydratase (Iga16) were shown to catalyze the reduction of a b-
keto group and dehydration of a b-hydroxy group, respectively, to
form a trans double bond. Incubation of the acyl carrier protein
respond to PR or NR PKSs. Meanwhile, a phylogenetically new
subfamily of type II PKSs that should be involved in the for-
mation of polyene structure have been discovered in the past
few years (Figures S1 and S2). Examples are IgaPKS in ishi-
gamide biosynthesis (Scheme 1) from Streptomyces sp.
[
8]
MSC090213JE08 , Sky PKS in skyllamycin biosynthesis from
[9]
Streptomyces sp. Acta 2897 and Sim/Smc PKS in simocy-
(
Iga10), the ketosynthase-chain length factor complex (Iga11-
clinone biosynthesis from Streptomyces antibioticus and Kita-
Iga12), Iga13 and Iga16 with malonyl and hexanoyl-CoAs and
NADPH followed by KOH hydrolysis resulted in the formation of
four unsaturated carboxylic acids (C , C10, C12 and C14), indicating
8
that IgaPKS catalyzes tetraene formation by repeating the cycle of
condensation, keto-reduction and dehydration with strict stereo-
specificity. We propose “highly reducing type II PKS subfamily” for
the polyene-producing type II PKSs.
[
10]
satospora sp. 152608 . Interestingly, these type II PKSs are
distributed only in actinobacteria (Figure S3). Although these
putative polyene-producing type II PKSs seem to correspond to
HR PKSs, none of them have been characterized in vitro so far.
IgaPKS was predicted to assemble the polyene portion of
ishigamide by a cycle of condensation, reduction and dehydra-
tion, catalyzed by KS-CLF (Iga11-Iga12), KR (Iga13) and DH
[
8]
(
Iga16), respectively (Scheme 1) . The polyketide chain elon-
Polyketide synthases (PKSs) are enzymes responsible for
the biosynthesis of structurally diverse and pharmaceutically
useful secondary metabolites, polyketides. Generally, PKSs are
gation occurs on ACP (Iga10). To analyze the function of these
Iga proteins, we obtained them as recombinant proteins (Figure
S4a and b and Figure S5). Iga10 was co-expressed with a
[
1]
[
11]
classified into three major types . Type I PKSs are megasyn-
thases composed of multiple functional domains, including ke-
tosynthase (KS), acyl transferase (AT), acyl carrier protein
phosphopantetheinyl transferase (Sfp)
to generate holo-
Iga10 (4’-phosphopantetheinylated form). Almost all of the puri-
fied Iga10 took the form of holo-Iga10 as determined by LC-MS
analysis (Figure S6). Interestingly, Iga11 and Iga12 could not
be obtained as a soluble protein individually. Therefore, we co-
expressed His-tagged Iga11 with Strep-tagged Iga12. Purifica-
(
ACP), and possibly ketoreductase (KR), dehydratase (DH) and
enoyl reductase (ER). They can be further divided into modular
and iterative type I PKSs. Iterative type I PKSs can be further
classified into three subtypes, highly reducing (HR), partially
reducing (PR) and non-reducing (NR) PKSs, according to the
2
+
tion with Ni -NTA affinity chromatography followed by strep-
tactin affinity chromatography gave the mixture of purified Iga11
and Iga12 (Figure S5). Thus, Iga11 and Iga12 were shown to
form a complex.
[
1,2]
presence of KR, DH and ER . Among these subtypes, HR
iterative type I PKSs are known for synthesizing highly reduced
[
3]
[4]
polyunsaturated fatty acids (PUFAs) , enediynes
and
[
5]
statins . Modular type I PKSs are also capable of producing
highly reduced polyketides, such as macrolide polyene antibiot-
Iga11
Iga12
S CoA
R
S Iga10
Iga13
[
6]
O
O
O
ics . In contrast, type II PKSs are complexes of discrete en-
[
7]
zymes known for biosynthesizing aromatic compounds . In a
typical type II PKS system, a KS-chain length factor (CLF) het-
erodimer iteratively elongates a poly-b-ketone chain linked to
Iga11
Iga12
H
S Iga10
Iga10
malonyl-Iga10
OH O
[
7]
an ACP . The full-length poly-b-ketone chain is then subjected
Iga16
to accessory enzymes such as cyclase (CYC) and aromatase
R
S Iga10
malonyl-CoA
(
ARO), resulting in aromatic polyketides. In some cases, a keto
O
reduction catalyzed by KR takes place after polyketide elonga-
tion to generate aromatic polyketides with fewer hydroxy
groups. Thus, aromatic polyketide-producing type II PKSs cor-
S Iga10
O
[
a]
b]
Danyao Du, Dr. Yohei Katsuyama, Dr. Yasuo Ohnishi
Department of Biotechnology, Graduate School of Agricultural and
Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, To-
kyo 113-8657, Japan
H
N
OH
OH
O
O
E-mail: *Y.O.; ayasuo@mail.ecc.u-tokyo.ac.jp
ishigamide
*Y. K.; aykatsu@mail.ecc.u-tokyo.ac.jp
[
Dr. Kazuo Shin-ya
National Institute of Advanced Industrial Science and Technology
Scheme 1. Predicted biosynthetic pathway for ishigamide.
(
AIST), 2-4-7, Aomi, Koto-ku, Tokyo 135-0064, Japan
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
Alignment of Iga13 with other KRs revealed that the canoni-
of b-hydroxy intermediates synthesized by Iga13 (Scheme 1).
As expected, Iga16 converted 2R to trans-2-octenoyl-NAC (3)
in vitro (Figure 1a and c and Figures S10 and S11). This result
is consistent with the empirical rule proposed among type I
[
12]
cal catalytic tetrad (Ser-Tyr-Lys-Asn) of KR and the NAD(P)-
binding site are conserved in Iga13 (Figure S7). However,
Iga13 lacks any motifs that were reported to determine the ste-
[
13]
[13]
reo-specificity of KRs (LDD motif in type I PKSs
and PGG
PKSs that R-alcohols provide E-olefins . In contrast, Iga16 did
[
14]
motif in type II PKSs ). We examined the activity of Iga13
not recognize 2S as its substrate, showing that Iga16 has strict
stereo-specificity towards R-configured hydroxy group (Figure
S10). Iga16 also catalyzed stereo-selective hydration of 3 to
form 2R (Figure 1a and c and Figure S10). This result explains
the incomplete conversion of 2R to 3 in vitro; the ratio of 2R to
3 was approximately 1:1.2 when the reaction reached equilibri-
um. During ishigamide biosynthesis, the dehydration reaction is
probably driven by the following condensation reaction.
toward 3-oxooctanoyl-N-acetylcysteamine (NAC) (1) (Figure
1
a). Incubation of 1 with NADPH and Iga13 led to the formation
of (3R)-hydroxyoctanoyl-NAC (2R) (Figure 1b and Figure S8).
Formation of (3S)-hydroxyoctanoyl-NAC (2S) was not observed,
indicating that Iga13 exhibits strict stereo-specificity. The Mich-
aelis–Menten kinetic parameters were determined; K
m
= 0.29 ±
.08 mM, kcat = 59.2 ± 5.6 min for 1 (Figure S9). The kinetic
parameters are comparable to those reported for the KRs from
-
1
0
[
19]
Similar to typical type II PKS ACPs , Iga10 displayed self-
acylation activity when incubated with a high concentration of
malonyl-CoA. Incubation of 100 µM holo-Iga10 with 1 mM mal-
onyl-CoA at 30°C for 4 h converted half of the holo-Iga10 to
malonyl-Iga10 (Figure S12). To improve the efficiency of ACP
acylation, a malonyl-CoA: acyl carrier protein transacylase
[
15]
type I (LovB in lovastatin biosynthesis ) and type II (ActKR in
[
16]
actinorhodin biosynthesis ) PKS systems (Table S1). Iga13
also accepted NADH as a cofactor (Figure S8). However, the
velocity of reduction of 1 using NADH is only 2.3% of that using
NADPH.
[
20]
In silico analysis suggested that Iga16 possesses a hotdog
(MAT) from Streptomyces coelicolor A3(2) was also prepared
as a C-terminally His-tagged recombinant protein (Figure S4c)
and applied to the in vitro assay. However, addition of MAT did
not significantly improve the acylation efficiency (data not
shown).
[
17]
[18]
fold and shows similarity to NodN and MaoC dehydratases.
Although Iga16 shows low amino acid sequence similarities to
other characterized DHs from fatty acid synthase and PKS, we
assumed that Iga16 should be responsible for the dehydration
Figure 1. In vitro analysis of Iga13 and Iga16 using NAC substrate analogues that mimic the polyketide intermediates attached to ACP. (a) Schemes of Iga13 and
Iga16 reactions. (b) HPLC analysis of Iga13 reaction. Incubation of 1 with active Iga13 led to the formation of 2R. (c) HPLC analysis of Iga16 reaction. Incubation
of 2R with active Iga16 led to the formation of 3, while incubation of 3 with active Iga16 led to the formation of 2R.
[
21]
The catalytic triad (Cys-His-His) of KS
is conserved in
keto-group of the polyketide intermediate to a double bond.
Therefore, we attempted to reconstruct the polyene biosynthe-
sis by incubating all IgaPKS components (Iga10, Iga11-Iga12,
Iga13 and Iga16) with malonyl-CoA, hexanoyl-CoA and NADPH.
By LC-MS analysis of the polyketide intermediates removed
from Iga10 by KOH hydrolysis, we detected the formation of
four unsaturated carboxylic acids: (2E)-octenoic acid (4),
(2E,4E)-deca-2,4-dienoic acid (5), (2E,4E,6E)-dodeca-2,4,6-
trienoic acid (6) and (2E,4E,6E,8E)-tetradeca-2,4,6,8-tetraenoic
acid (7) (Figure 2 and Figure S16). Formation of these unsatu-
rated carboxylic acids was observed only when all of the Iga
proteins were included in the reaction system. Also, malonyl-
CoA and hexanoyl-CoA were both required for the reaction.
These results clearly indicate that IgaPKSs accomplish the
biosynthesis of polyene structure by continuous reaction cycles
of condensation, reduction and dehydration.
[
22,23]
Iga11, while the conserved triad (Gln-Asp-Arg) of CLF
is
converted to Arg-Thr-Asp in Iga12 (Figure S13). To investigate
the catalytic activity of Iga11-Iga12, Iga11-Iga12 and Iga10
were incubated with malonyl-CoA and hexanoyl-CoA. After
incubation, the reaction mixture was treated with KOH to hydro-
lyze the growing polyketide intermediates off from Iga10. How-
ever, we could not observe formation of any detectable prod-
ucts (Figure S14). Therefore, we turned to LC-MS analysis of
Iga10 to examine the modification of Iga10 in the presence of
Iga11-Iga12. As a result, we detected the formation of a trace
amount of 3-oxooctanoyl-Iga10 (Figure S15), indicating that the
Iga11-Iga12 complex could catalyze the decarboxylative con-
densation between hexanoyl-CoA (starter unit) and malonyl-
Iga10 (extender substrate).
This result also implies that Iga11-Iga12 cannot catalyze a
further condensation unless Iga13 and Iga16 convert the b-
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Angewandte Chemie International Edition
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COMMUNICATION
tide chain. We speculate that Iga11-Iga12 may form a narrower
but more accessible reaction chamber compared with the KS-
CLF of typical type II PKS. When the alkaline hydrolysis proce-
dure was skipped, the formation of unsaturated carboxylic acids
could no longer be observed (Figure 2). This result suggests
that IgaPKS cannot hydrolyze the full-length polyketide chain
off from Iga10. We believe that the release of polyketide chain
is accomplished by the attack of b-alanine, which should be
catalyzed by an unknown enzyme in the ishigamide-producing
strain.
The reaction cycle catalyzed by Iga11-Iga12, Iga13 and
[
2,26]
Iga16 resembles that of HR iterative type I PKSs
. In the
iterative type I PKS system, polyketide intermediates are prob-
ably accommodated in the hydrophobic reaction chamber as
[
27]
reported in the modular PKS system . In contrast, in the
IgaPKS system, the growing polyketide intermediates need to
be transported from one catalytic domain to a discrete domain
frequently and efficiently. We speculate that Iga10 plays an
important role in protecting the unstable polyketide intermedi-
ates as reported in canonical ACPs in the type II PKS sys-
[
28,29]
tem
. Iga10 also seems to deliver the growing polyketide
intermediates only to the Iga enzymes that catalyze the follow-
ing reactions. The delivery seems to be well controlled, be-
cause no skipping of the modification steps took place. We
speculate that the interaction between the polyketide interme-
diate-tethered Iga10 and each catalytic domain should be criti-
cal for such “traffic control”.
In summary, we confirmed the enzymatic activity of each
IgaPKS protein and successfully reconstituted IgaPKS capable
of catalyzing polyene formation in vitro. As mentioned above,
while type I PKSs can biosynthesize highly reduced, partly re-
duced and non-reduced polyketides, type II PKSs have long
been considered to be used for the biosynthesis of aromatic
compounds, which correspond to the partly reduced and non-
Figure 2. LC-MS analysis of in vitro IgaPKS reaction synthesizing the poly-
ene portion of ishigamide. (a) Scheme of the in vitro reconstitution of the
IgaPKS system. (b) The reaction mixture was applied to TCA precipitation
and KOH hydrolysis. The solution was then extracted with ethyl acetate after
acidification and applied to LC-MS analysis. The compounds indicated by
asterisks seem to be generated by cis-trans isomerization of one or more
double bonds of 7, because these compounds and 7 showed very similar UV
absorbance spectra. This isomerization should be due to the instability of the
[7]
reduced polyketides . Our study demonstrated that type II
PKSs also possess the ability to biosynthesize highly reduced
polyketides, thus expanding our knowledge of the product vari-
ety of type II PKS. As with the type I PKS system, we propose
that IgaPKS serve as the representative of the HR type II PKS
subfamily. We speculate that coevolution of KS-CLF, ACP and
their accessory enzymes resulted in the generation of HR type
II PKSs. We believe that further studies on IgaPKS, such as the
crystal structure analysis of each component of IgaPKS, will
shed light on the biosynthetic logic of the HR type II PKS sys-
tem.
[
24]
conjugated-double bonds
by acidic and alkaline conditions, heating, or
light exposure. The peak at 15 min seems to be derived from degradation of
CoA or NADPH by the KOH hydrolysis.
In this polyene biosynthesis reconstitution system, no car-
boxylic acids with b-keto-group or b-hydroxy group were formed,
suggesting that the condensation reaction catalyzed by Iga11-
Iga12 is the rate-limiting step. Furthermore, no carboxylic acids
with chain length exceeding 14 were detected, suggesting the
chain length of the final polyketide product is strictly controlled.
In a typical type II PKS system, the length of polyketide chain is
determined by the length of amphipathic tunnel that forms at
Acknowledgments
[
25]
This research was supported in part by a Grant-in-Aid for Sci-
entific Research on Innovative Areas from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of Japan
(MEXT) (JP25108706 and JP17H05432), Grant-in-Aid for Ja-
pan Society for the Promotion of Science (JSPS) Research
Fellow (JP17J09439) and JSPS A3 Foresight Program.
the KS-CLF heterodimer interface . A similar mechanism may
be applicable to the Iga11-Iga12 complex. However, different
from the KS-CLF of typical type II PKS that accommodates a
growing highly flexible polyketone chain within its reaction tun-
nel, Iga11-Iga12 seems to recognize a rigid unsaturated acyl
chain as its substrate and allow the growing polyketide inter-
mediate to enter and exit its reaction tunnel frequently. Fur-
thermore, while typical type II PKSs commonly produce C16-C30
polyketide chains, Iga11-Iga12 produces a shorter C14 polyke-
Keywords: biosynthesis • polyene • polyketides • type II
polyketide synthase • Streptomyces
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Angewandte Chemie International Edition
10.1002/anie.201709636
COMMUNICATION
While type II polyketide synthases
Danyao Du, Dr. Yohei Katsuyama*, Dr.
Kazuo Shin-ya, Dr. Yasuo Ohnishi*
malonyl-ACP
hexanoyl-CoA
(
PKSs) are known for producing aro-
. CLF
KS
matic compounds, in vitro analysis of
the type II PKS for ishigamide biosyn-
thesis demonstrated that it catalyzes
tetraene formation by repeating the
cycle of condensation, keto-reduction
and dehydration with strict stereo-
specificity. This study expands our
knowledge of the catalytic property of
type II PKS and establishes a subfam-
ily of type II PKS, highly reducing type
II PKS, which synthesizes polyenes.
ACP
malonyl-CoA
Page No. – Page No.
ACP
ACP
S
O
Reconstitution of a type II polyketide
synthase catalyzing polyene for-
mation
S
S
O
O
O
R
R
ACP
KR
DH
S
O
HO
5
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