A type III polyketide synthase from Rhizobium etli condenses malonyl CoAs to a heptaketide pyrone with unusually high catalytic efficiency

Article abstract of DOI:10.1039/c2mb25347j

A novel type III polyketide synthase (RePKS) from Rhizobium etli produced a heptaketide pyrone using acetyl-CoA and six molecules of malonyl-CoA. Its catalytic efficiency (k_cat/K_m = 5230 mM^-1 min^-1) for malonyl CoA was found to be the highest ever reported. Molecular dynamics studies revealed the unique features of RePKS. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2mb25347j

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A type III polyketide synthase from Rhizobium etli condenses malonyl
CoAs to a heptaketide pyrone with unusually high catalytic efficiencyw
Marimuthu Jeya,a Tae-Su Kim,a Manish Kumar Tiwari,a Jinglin Li,a Huimin Zhao*bcd and
Jung-Kul Lee*a
Received 28th August 2012, Accepted 28th September 2012
DOI: 10.1039/c2mb25347j
A novel type III polyketide synthase (RePKS) from Rhizobium
etli produced a heptaketide pyrone using acetyl-CoA and six
being a type III PKS (Fig. S1, ESIw). The RePKS encoding
gene was cloned and expressed in E. coli BL21-CodonPlus
(DE3)-RIL with an N-terminal 6ÂHis affinity tag. The purified
His6-tagged RePKS protein gave a single protein band at B37 kDa
on SDS-PAGE. RePKS efficiently accepted malonyl-CoA as a
sole substrate and yielded a single product with a parent ion
peak [M + H]+ at m/z 277 on LC/ESI-MS (Fig. 1). The
molecular weight of the product corresponded to a heptaketide
pyrone. A heptaketide pyrone was also detected in the reaction
products of Rheum palmatum aloesone synthase and Aloe
arborescens PKS3 which produced aloesone as a final product.5
Hence, the product was further confirmed by the instrumental
analysis as described previously.5 In the reaction of RePKS with
malonyl-CoA, a major product had a retention time of 14.7 min
(Fig. 1) in HPLC that was similar to the minor heptaketide
pyrone intermediate produced by Aloe arborescens PKS3.
Instrumental analysis (HPLC, UV, MS and HRMS) of the
product obtained by a large-scale enzyme reaction confirmed
the product to be 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-
oxoethyl)-4-hydroxy-2-pyrone. It was found that acetyl-CoA
resulting from decarboxylation of malonyl-CoA was a better
starter substrate than malonyl-CoA for RePKS. The substrate
preference was confirmed by using labeled 14C malonyl-CoA
molecules of malonyl-CoA. Its catalytic efficiency (kcat/Km
=
5230 mMÀ1 minÀ1) for malonyl CoA was found to be the highest
ever reported. Molecular dynamics studies revealed the unique
features of RePKS.
Type III polyketide synthases (PKSs) generate the backbones of a
variety of plant secondary metabolites including chalcones,
stilbenes, phloroglucinols, resorcinols, benzophenones, biphenyls,
bibenzyls, chromones, acridones, pyrones, and curcuminoids.1,2
The plant type III PKSs share 30–95% amino acid sequence
identity with each other, but only 21–31% identity with those of
bacterial origin. The functional diversity of the type III PKSs is
attributable to differences in their selection of the starter substrate,
the number of polyketide chain extensions, and the mechanisms
of their cyclization reactions.
Several bacterial type III PKSs have been characterized at
present; for example, RppA from Streptomyces coelicolor and
Streptomyces griseus3 and PhlD from Pseudomonas fluorescens.4
This work reports the characterization of the gene product
(RePKS) of AAM55027.1 from Rhizobium etli. The RePKS is a
distinct type III PKS that catalyzes the synthesis of a heptaketide
pyrone using acetyl-CoA and six molecules of malonyl-CoA. This
is the first report that describes the catalytic properties of a type
III PKS from R. etli.
Results
The amino acid alignment of RePKS with plant, bacterial, and
fungal type III PKSs showed conservation of the Cys-His-Asn
catalytic triad in RePKS, thus providing evidence for RePKS
a Department of Chemical Engineering, Konkuk University,
Seoul 143-701, Korea. E-mail: jkrhee@konkuk.ac.kr;
Fax: +82-2-458-3504
b Departments of Chemical and Biomolecular Engineering, Chemistry,
and Bioengineering, University of Illinois, 600 South Mathews
Avenue, Urbana, IL 61801, USA. E-mail: zhao5@illinois.edu;
Fax: +1 217-333-5052
Fig. 1 HPLC analysis of the product synthesized by RePKS with
malonyl CoA as a starter and extender substrate. The inset figure
shows the ESI-MS chromatogram and structure of the product peak at
14.7 min which corresponds to a heptaketide pyrone (MW = 276).
The reaction product of RePKS was analyzed by LC/ESI-MS in
positive mode.
c Institute for Genomic Biology, University of Illinois,
600 South Mathews Avenue, Urbana, IL 61801, USA
d Center for Biophysics and Computational Biology, University of
Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2mb25347j
c
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Table 1 Relative activity of the RePKS protein with different starter
units. Results are means (n = 3) with SE values less than 15%
Starter CoA
Incorporation efficiency by RePKS (%)
Acetyl-CoA (2a)
100
56.7
5.8
13.6
4.7
Malonyl-CoA (1a)
Hexanoyl-CoA (2b)
Lauroyl-CoA (2d)
Stearoyl-CoA (2g)
Benzoyl-CoA (2h)
6.2
and acetyl-CoA. Reaction was performed by the addition of
RePKS to [2-14C]malonyl-CoA in the presence and absence of
acetyl-CoA. Approximately a two-fold increase in the yield
of the heptaketide was observed when acetyl-CoA was present
in the reaction mixture. The substrate specificity of RePKS
was further analyzed using CoA esters of C2 to C18 straight-
chain fatty acids as starter substrates, with malonyl-CoA as the
extender. The incorporation efficiency of each acyl-CoA is listed
in Table 1. Use of butyryl CoA as the starter substrate resulted
in a heptaketide and also a small amount of a product that was
identified by LC/ESI-MS to be a triketide (Mr = 154). With
hexanoyl-CoA as the starter, RePKS produced a triketide (3b)
(Mr = 182) as determined by LC/ESI-MS.
Similarly, use of octanoyl-CoA as the starter generated a
triketide (Mr = 210) product. Decanoyl-CoA was also accepted
by RePKS and resulted in the formation of a triketide (3c)
(Mr = 238) and a tetraketide (4c) (Mr = 280). Further, RePKS
showed activity with long chain acyl-CoAs including lauroyl-
CoA (2d), myristoyl-CoA (2e), palmitoyl-CoA (2f) and
stearoyl-CoA (2g), and aromatic substrate benzoyl-CoA
(2h). The heptaketide pyrone was detected in all reactions
with various starter-CoAs along with new products, and the
molecular formulae of products were confirmed by HRMS.
ESI-MS data are provided in the ESI.w The product formation
by RePKS and the general structures of all the products are
given in Fig. 2.
Fig. 2 Summary of RePKS reactions with various acyl-CoA starter
substrates. (A) Heptaketide pyrone formation by RePKS with malonyl-
CoA as a starter and extender substrate. (B) RePKS with various
other acyl-CoAs as starter substrates produces corresponding tri and
tetraketide pyrone products.
molecules of malonyl-CoA, a homology model of RePKS11
was built based on the crystal structure of THNS from
S. coelicolor (Protein Data Bank accession code 1U0M) which
shares 32% sequence identity with RePKS. THNS accepts
only malonyl-CoA, not acetyl-CoA. Prior biochemical and
structural studies of plant and bacterial type III PKSs have
established the importance of several residues near the active
site in controlling substrate and product specificities. These
include Thr-197, Gly-256, and Ser-338 of M. sativa CHS.12
Thr-197 of CHS (or Cys-171 of THNS) is replaced by Ala-175
in RePKS (Fig. 3). The cavity volume of RePKS is larger
(756 A3) than that of THNS (622 A3), which can probably
explain that RePKS performs condensations of six molecules
of malonyl-CoA compared to the utilization of five malonyl
CoAs by THNS (Fig. S2, ESIw). The homology model of
R. palmatum aloesone synthase (ALS) had a much larger
cavity volume (1170 A3) than that of THNS (622 A3) and
thus it produced aloesone after condensation with seven
malonyl-CoAs.5 Further, the size of the active site cavity
physically limits the number of malonyl-CoA condensations,
as implied from the X-ray crystal structures of 2-pyrone
synthase and CHS.13,14
RePKS showed the highest activity towards malonyl-CoA.
RppA from S. griseus, apart from using five molecules of
malonyl-CoA as a starter to produce THN, accepted aliphatic
acyl-CoAs with the carbon lengths from C4 to C8 as starter
substrates and catalyzed sequential condensation of malonyl-
CoA to yield a-pyrones and phloroglucinols.6 PhlD from
P. fluorescens produces phloroglucinol from three molecules
of malonyl-CoA and also showed broad substrate specificity
by accepting C4–C12 aliphatic acyl CoAs.4 RePKS differs
from these enzymes in carrying out more condensations, i.e.,
condensations of six molecules of malonyl-CoA with acetyl-
CoA to produce a heptaketide pyrone.
Kinetic parameters were determined according to Zha et al.4
Under the optimal assay conditions (30 1C, pH 8.0), the
apparent kcat and Km values of purified RePKS for malonyl-
CoA were 22.5 minÀ1 and 4.3 mM, respectively, giving a
catalytic efficiency (kcat/Km) of 5230 mMÀ1 minÀ1. The Km
value of RePKS is within the range of those reported for other
type III PKSs.5,7–10 However, the kcat/Km value of RePKS
towards malonyl-CoA was the highest among type III PKSs
reported and 2.7 fold higher than that of PhlD.
The RePKS enzyme showed highest catalytic efficiency
towards malonyl-CoA than other reported type III PKSs
(Table 2). The structures of CHS complexed with CoA,
acetyl-CoA, and hexanoyl-CoA defined the overall placement
of CoA in the enzyme.13,14 Because Cys-164 is the catalytic
To investigate the structural basis for the ability of RePKS
to accept acetyl-CoA and perform condensations with six
c
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Table 2 Comparison of kinetic constants of malonyl CoA determined for type III PKSs
Source
kcat (minÀ1
)
kcat/Km (mMÀ1 minÀ1
)
Ref.
Heptaketide-ALS (Rheum palmatum)
PCS (Aloe arborescens)
OKS (Aloe arborescens)
0.027
0.445
0.094
0.430
NR
NR
0.007
24
0.31
6.3
7
15
16
9
8
10
5
4
6
17
0.98
69.35
NR
NR
0.085
1883
828
BIS (Sorbus aucuparia)
BPS (Hypericum androsaemum)
DluHKS (Drosophyllum lusitanicum)
PKS3 (Aloe arborescens)
PhlD (Pseudomonas fluorescens)
RppA (Streptomyces griseus)
DpgA (Amycolatopsis orientalis)
RePKS (Rhizobium etli)
0.770
0.810
22.5
54
5230
This study
must be accessible to the cysteine Sg. Interaction between the
thioester carbonyl oxygen and the side-chains of Asn-336 and
His-303 of CHS facilitates substrate binding and stabilizes the
transition state during nucleophilic attack of Cys-164 on the
thioester carbonyl.13,14 With these constraints, we modeled
the binding of malonyl-CoA to both the RePKS modeled
structure and THNS structure (Fig. 3). When malonyl-CoA
was docked into the CoA binding site of RePKS, malonyl-CoA
was bound through hydrogen bonds (green dotted lines) with
Asn-308 (2.4 A). An interacting distance of 3.6 A has been
observed between the thioester carbonyl oxygen and nitrogen
(Ne) atom of His-275. While a distance of 3.0 A was observed
between the sulphur of the catalytic cysteine and the thioester
carbonyl group of malonyl-CoA. Other potential hydrogen
bond interactions were also observed with Ser-48 (2.9 A)
(Fig. 3A). However, when malonyl-CoA was docked into the
CoA binding site of THNS, there were no hydrogen bonding
interactions between malonyl CoA and enzyme. Malonyl-CoA
showed interacting distances of 3.4 A, 3.9 A and 3.0 A
with Asn-303, His-270, and Cys-138, respectively (Fig. 3B).
Moreover, superimposition of the RePKS–malonyl–CoA
complex and the THNS–malonyl–CoA complex (Fig. S3, ESIw)
clearly defined the differences in amino acids (Trp228/Tyr224
and Ala175/Cys171) in the active site pocket and amino acid
side chains (Gln-241/Lys-237; Lys-280/Arg-275; Ser-48/Asn-44)
in the CoA binding site between these two enzymes.
To investigate the role of the residues (Gln-241, Lys-280,
and Ser-48) in catalytic efficiency, they were each mutated to
Ala. The recombinant enzymes carrying K280A, Q241A, and
S48A mutations were expressed and purified. Their activity
with malonyl-CoA was measured and compared with that of
the wild-type RePKS. The kcat/Km value of the mutants was
decreased from 50 to 72-fold compared to that of the wild type
RePKS (Table S1, ESIw), suggesting that all of these residues
play a significant role in the turnover of malonyl-CoA. The
role of these residues in catalysis will be further investigated by
site-directed mutagenesis.
Fig. 3 Substrate docking of RePKS and THNS with malonyl CoA in
the CoA binding tunnel. (A) Malonyl-CoA docking into the CoA
binding tunnel of RePKS. Malonyl-CoA was bound through hydro-
gen bonds (green dotted lines) with Asn-308 (2.4 A). Right figure
represents the CoA binding tunnel of the modeled RePKS. Amino acid
residues are shown in a stick model, residues in the CoA binding
tunnel are colored with elemental carbon while catalytic amino acid
residues are shown with blue color carbon. Malonyl-CoA is repre-
sented in a ball and stick model with green color carbon. (B) Malonyl-
CoA docking into the CoA binding tunnel of THNS. Malonyl-CoA
showed interacting distances of 3.4 A, 3.9 A and 3.0 A with Asn-303,
His-270, and Cys-138, respectively. Right figure represents the CoA
binding tunnel of THNS. Amino acid residues are shown in a stick
model, residues in the CoA binding tunnel are colored with elemental
carbon while catalytic amino acid residues are shown with black color
carbon. Malonyl-CoA is represented in a ball and stick model with
black color carbon.
Conclusions
RePKS characterized in the present study produced a heptaketide
pyrone product from acetyl-CoA and malonyl-CoA. Although
RePKS displayed broad substrate specificity, it exhibited the
highest catalytic efficiency towards malonyl-CoA among type
III PKSs reported. Overexpression of RePKS in engineered
E. coli strain (with the improved cellular malonyl-CoA level)
nucleophile required for tethering of the starter group and the
growing polyketide chain, the CoA-thioester carbonyl carbon
c
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will be useful for biosynthesis of polyketide products in higher
yields. Crystal structure analyses combined with mutational studies
are now in progress to probe the structural basis for the starter
substrate specificity and the catalytic properties of RePKS.
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(220-2009-1-D00033 and 2012M3C5A1053339).
6 N. Funa, Y. Ohnishi, Y. Ebizuka and S. Horinouchi, J. Biol.
Chem., 2002, 277, 4628–4635.
7 I. Abe, Y. Utsumi, S. Oguro and H. Noguchi, FEBS. Lett., 2004,
562, 171–176.
8 B. Liu, H. Falkenstein-Paul, W. Schmidt and L. Beerhues, Plant J.,
2003, 34, 847–855.
9 B. Liu, T. Raeth, T. Beuerle and L. Beerhues, Planta, 2007, 225,
1495–1503.
10 A. Jindaprasert, K. Springob, J. Schmidt, W. De-Eknamkul and
T. M. Kutchan, Phytochemistry, 2008, 69, 3043–3053.
11 M. Tiwari and J. K. Lee, J. Mol. Graphics Modell., 2010, 28,
707–713.
Notes and references
1 M. A. Fischbach and C. T. Walsh, Chem. Rev., 2006, 106,
3468–3496.
2 J. Staunton and K. J. Weissman, Nat. Prod. Rep., 2001, 18,
380–416.
12 M. B. Austin and J. P. Noel, Nat. Prod. Rep., 2003, 20, 79–110.
13 J. L. Ferrer, J. M. Jez, M. E. Bowman, R. A. Dixon and J. P. Noel,
Nat. Struct. Mol. Biol., 1999, 6, 775–784.
14 J. M. Jez, M. B. Austin, J.-L. Ferrer, M. E. Bowman, J. Schroder
¨
3 M. Izumikawa, P. R. Shipley, J. N. Hopke, T. O’Hare, L. Xiang,
J. P. Noel and B. S. Moore, J. Ind. Microbiol. Biotechnol., 2003, 30,
510–515.
and J. P. Noel, Chem. Biol., 2000, 7, 919–930.
15 I. Abe, Y. Utsumi, S. Oguro, H. Morita, Y. Sano and H. Noguchi,
J. Am. Chem. Soc., 2005, 127, 1362–1363.
4 W. Zha, S. B. Rubin-Pitel and H. Zhao, J. Biol. Chem., 2006, 281,
32036–32047.
16 I. Abe, S. Oguro, Y. Utsumi, Y. Sano and H. Noguchi, J. Am.
Chem. Soc., 2005, 127, 12709–12716.
5 Y. Mizuuchi, S.-P. Shi, K. Wanibuchi, A. Kojima, H. Morita,
H. Noguchi and I. Abe, FEBS. J., 2009, 276, 2391–2401.
17 C. C. Tseng, S. M. McLoughlin, N. L. Kelleher and C. T. Walsh,
Biochemistry, 2004, 43, 970–980.
c
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