Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum

Article abstract of DOI:10.1016/j.phytochem.2008.03.013

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.

Full text of DOI:10.1016/j.phytochem.2008.03.013

Phytochemistry 69 (2008) 3043–3053
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Pyrone polyketides synthesized by a type III polyketide synthase
from Drosophyllum lusitanicum
c
b
Aphacha Jindaprasert a,b,c, Karin Springob c,d, , Jürgen Schmidt , Wanchai De-Eknamkul ,
*
Toni M. Kutchan c,d
a Faculty of Agroindustry, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
b Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
c Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, 06120 Halle, Saale, Germany
d Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2007
Received in revised form 11 February 2008
Available online 6 May 2008
To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusit-
anicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a
cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to
known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The trans-
lated polypeptide sequence of DluHKS showed 51–67% identity with other plant type III PKSs. Recombi-
nant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out
sequential decarboxylative condensations with malonyl-CoA yielding a-pyrones from three to six acetate
units. However, naphthalenes, the expected products, were not isolated. Since the main compound pro-
duced by DluHKS is a hexaketide a-pyrone, and the naphthoquinones in D. lusitanicum are composed of
six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An
involvement of accessory proteins in this biosynthetic pathway is discussed.
Keywords:
Drosophyllum lusitanicum link
Drosophyllaceae
Naphthoquinone
Hexaketide synthase
Pyrone synthase
Type III polyketide synthase
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
(Likhitwitayawuid et al., 1998), and anticancer (Sugie et al.,
1998; Sandur et al., 2006) effects.
Drosophyllum lusitanicum Link, the dewy pine, is a carnivorous
plant that occurs in the southern part of the Iberian peninsula
and northern Morocco. Taxonomically, D. lusitanicum belongs to
the monotypic family Drosophyllaceae, as shown by recent molec-
ular studies, but is closely related to other families with carnivo-
rous plants, e.g the Droseraceae, Nepenthaceae and
Dioncophyllaceae (Meimberg et al., 2000; Heubl et al., 2006). A
chemotaxonomic marker of these taxa and related families with-
out carnivory, e.g. the Plumbaginaceae, is the occurrence of aceto-
genic naphthoquinones. These secondary metabolites serve as
defense compounds (Peters et al., 1995; Bringmann et al., 1999)
and as antifeedants against insects (Tokunaga et al., 2004a,b), but
some have allelopathic effects, as well (Dornbos and Spencer,
1990; Higa et al., 1998). Naphthoquinones are also known to pos-
sess useful pharmacological activities. Plumbagin (8) (2-methyl-5-
hydroxy-1,4-naphthoquinone), for example, was shown to have
antimicrobial (Durga et al., 1990; Didry et al., 1994), antimalarial
The biosynthesis of naphthoquinones can occur via three differ-
ent routes in higher plants. They can be produced from geranylated
p-hydroxybenzoic acid, as in the case of alkannin and shikonin
(Schmid and Zenk, 1971; Inouye et al., 1979), or from iso-chorismic
acid and a-ketoglutaric acid via o-succinylbenzoic acid, providing
the precursor of vitamin K (Leistner, 1999). A third pathway lead-
ing to the naphthoquinones plumbagin (8), droserone and 7-meth-
yljuglone utilizes acetate precursors. First evidence of this pathway
had been provided by tracer studies with Plumbago europaea, Dro-
sera species and Drosophyllum lusitanicum (Durand and Zenk,
1971,1974). Recent experiments by Bringmann and co-workers
confirmed the acetogenic origin of naphthoquinones in lianas of
the Ancistrocladaceae and Dioncophyllaceae families (Bringmann
et al., 2000). Based on these experiments, it was postulated that
plumbagin (8) and biosynthetically related naphthoquinones and
tetralones are synthesized by polyketide synthases (PKSs) via the
acetate-polymalonate pathway (Bringmann and Feineis, 2001).
All PKSs so far isolated from plants belong to the superfamily of
type III PKSs (Schröder, 2000; Austin and Noel, 2003), which com-
prises homodimeric proteins that catalyze the iterative decarboxy-
lative condensation of a starter coenzyme A (CoA) ester with
malonyl-CoA (1), or in some cases, methylmalonyl-CoA or ethylm-
alonyl-CoA (Schröder et al., 1998; Song et al., 2006). The best
* Corresponding author. Address: Donald Danforth Plant Science Center, 975
North Warson Road, St. Louis, MO 63132, USA. Tel.: +1 314 587 1490; fax: +1 314
587 1590.
E-mail address: kspringob@danforthcenter.org (K. Springob).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.03.013

3044
A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
Fig. 1. Postulated mechanism of plumbagin biosynthesis and comparison with the reaction of chalcone synthase (CHS). The PKS involved in plumbagin (8) biosynthesis
presumably catalyzes the decarboxylative condensation of acetyl-CoA (5) with five molecules of malonyl-CoA (1). The oxygen of the third acetate unit is probably removed by
a polyketide reductase (PKR) prior to the first cyclization, and one carbon is lost by decarboxylation.
Fig. 2. ClustalW alignment of DluHKS with other type III PKS amino acid sequences. Amino acids of the catalytic Cys-His-Asn triad are marked by triangles. Other amino acids
of the active site cavity, which are discussed in the text, are labeled by diamonds. Abbreviations: DluHKS, Drosophyllum lusitanicum HKS; Ghy2PS, Gerbera hybrida 2-pyrone
synthase; MsaCHS2, Medicago sativa CHS2; PinHKS, Plumbago indica HKS; RpaALS, Rheum palmatum aloesone synthase.

A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
3045
studied member of this family, chalcone synthase (CHS), is a type
III PKS that occurs ubiquitously in higher plants and catalyzes
the pivotal step of flavonoid biosynthesis by condensing a p-cou-
maroyl-CoA (2) starter with three molecules of malonyl-CoA (1)
(Fig. 1). The enzyme-linked intermediate tetraketide (3) subse-
quently undergoes an intramolecular Claisen condensation and
aromatization to yield naringenin chalcone (4). Apart from this
prominent example, more than 20 functionally distinct type III
PKSs from plants, fungi, and bacteria are known that produce a
large variety of natural products. This is achieved by variation of
starter and extender CoA esters, one to seven elongation steps with
malonyl-CoA (1) and different types of cyclization. Analysis of sev-
eral type III PKSs by X-ray diffraction and subsequent mutagenesis
studies provided evidence that substrate selectivity and chain
length depend mainly on the size and the shape of the active site
cavity (Ferrer et al., 1999; Jez et al., 2000a; Sankaranarayanan
et al., 2004). Many type III PKSs exhibit promiscuous substrate
specificities and accept a wide range of non-physiological sub-
strates in vitro (e.g. Abe et al., 2000, 2004a; Morita et al., 2000;
Samappito et al., 2002, 2003). In this case, however, mainly pyr-
one-type derailment products are formed, most probably due to
steric and/or electronic perturbations in the active site followed
by release of the uncyclized polyketide from the enzyme and spon-
taneous lactonization (Morita et al., 2000).
The PKS involved in plumbagin (8) biosynthesis has to catalyze
the condensation of six acetate units derived from acetyl-CoA (5)
and malonyl-CoA (1). The postulated intermediate hexaketide
undergoes two aldol cyclizations and is probably modified by a
ketoreductase (Fig. 1).
A recombinant type III hexaketide synthase of Plumbago indica,
PinHKS, recentlycharacterizedbyourgroup, catalyzestheformation
of a-pyrones from three to six acetate units (Springob et al., 2007).
Although the enzyme was able to condense six acetate units,
plumbagin (8) or a naphthalene derivative were not formed, and it
couldnot be unambiguouslyproven that thePKSis involvedin naph-
thoquinone biosynthesis in planta. In this paper, we report the isola-
tion of a similar cDNA from D. lusitanicum callus cultures by EST
sequencing termed DluHKS. The biochemical characterization of
the recombinant PKS provides additional evidence for the involve-
ment of hexaketide synthases in naphthoquinone biosynthesis.
Arachis hypogaea CHS
Medicago CHS2
0.1
98
76
Cassia alata CHS1
60
Hypericum androsaemum CHS
65
Hydrangea macrophylla CHS
Pinus strobus CHS
Pinus sylvestris CHS
100
95
plant
CHS
or STS
Pinus strobus STS1
Pinus sylvestris STS
100
Ruta graveolens CHS1
53
74
Vitis vinifera CHS
Hordeum vulgare CHS2
Hordeum vulgare CHS1
Zea mays CHS C2
Zea mays CHS WHP
Rheum tataricum STS
95
91
100
100
Rheum palmatum BAS
Hydrangea macrophylla STCS1
Hydrangea macrophylla CTAS
Ruta graveolens ACS1
100
Sorbus aucuparia BIS
Hypericum androsaemum BPS
61
plant
non CHS
or STS
Gerbera hybrida 2PS
DluHKS
54
Rheum ALS
PinHKS
70
100
Wachendorfia PKS
Aloe OKS
Aloe PCS
100
Aspergillus oryzae csyB
100
Aspergillus oryzae csyA
Fusarium graminearum FG08378.1
Neurospora crassa ORAS
100
fungi
100
73
Magnaporthe grisea MG04643.4
Streptomyces coelicolor THNS
Pseudomonas fluorescens PhlD
100
100
bacteria,
slime mold
Dictyostelium Steely2
82
Mycobacterium tuberculosis PKS18
E. coli FabH
Fig. 3. Relationship of DluHKS with other type III PKSs. The tree was constructed with protein sequences of type III PKSs from plants, fungi, bacteria and one slime mold PKS
(Steely 2). Except for ORAS of Neurospora crassa, the type III PKSs from fungi are not yet functionally characterized. E. coli b-ketoacyl synthase III (FabH) was used as outgroup.
The phylogenetic tree was calculated using TREECON (Van de Peer and De Wachter, 1994). Construction of the tree was performed using the Neighbour-Joining algorithm,
and bootstrap values higher than 50 are shown. Abbreviations: 2PS, 2-pyrone synthase; ACS, acridone synthase; ALS, aloesone synthase; BAS, benzalacetone synthase; BIS,
biphenyl synthase; BPS, benzophenone synthase; CHS, chalcone synthase; CTAS; p-coumaroyl triacetic acid lactone synthase; HKS, hexaketide synthase; OKS, octaketide
synthase; ORAS, 20-oxoalkylresorcylic acid synthase; PCS, pentaketide chromone synthase; STCS, stilbenecarboxylate synthase; STS, stilbene synthase; THNS, 1,3,6,8-tetra-
hydroxynaphthalene synthase. GenBank accession numbers: Aloe arborescens OKS (AAT48709) and PCS (AAX35541), Aspergillus oryzae csyA (BAD97390) and csyB (BA-
D97391), Arachis hypogaea CHS (AAO32821), Cassia alata CHS1 (AAM00230), Dictyostelium dioscoideum Steely2 (XP635518), E. coli FabH (1EBLB), Fusarium graminearum
FG08378.1 (XM388554), Gerbera hybrida 2-PS (P48391), Hordeum vulgare CHS1 (CAA41250) and CHS2 (CAA70435), Hydrangea macrophylla CHS (BAA32732), CTAS (BA-
A32733), and STCS1 (AAN76182), Hypericum androsaemum CHS (AAG30295) and BPS (AAL79808), Magnaporthe grisea MG04643.4 (XM362198), Medicago sativa CHS2
(P30074), Mycobacterium tuberculosis PKS18 (AAK45681), Neurospora crassa ORAS (XP960427), Pinus strobus CHS (CAA06077) and STS1 (CAA87012), Pinus sylvestris CHS
(CAA43166) and STS (AAB24341), Plumbago indica HKS (AB259100), Pseudomonas fluorescens PhlD (AAB48106), Rheum palmatum ALS (AAS87170) and BAS (AAK82824),
Rheum tataricum STS (AAP13782), Ruta graveolens ACS1 (S60241) and CHS1 (Q9FSB9), Sorbus aucuparia BIS (ABB89212), Streptomyces coelicolor THNS (NP625495), Vitis
vinifera CHS (BAA31259), Wachendorfia thyrsiflora PKS (AAY51378), Zea mays CHS C2 (AAW56964) and CHS WHP (CAA42763).

3046
A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
2. Results and discussion
To isolate the cDNAs encoding the naphthoquinone biosyn-
thetic enzymes, an expressed sequence tag (EST) analysis was per-
formed. A cDNA library was constructed with 2.5 lg Poly(A)+ RNA
isolated from D. lusitanicum calluses that have been shown to pro-
duce plumbagin (8) and drosophylloside, a naphthalenecarboxylic
acid glucoside (unpublished results). 2004 ESTs were obtained by
5’-sequencing of randomly chosen cDNA clones, and cluster analy-
sis yielded 1579 unigenes.
Two ESTs, W36 and Z25, showed sequence homology to known
chalcone synthase (CHS) superfamily members. Isolation of the
full-length clones was carried out by RACE PCR. The full-length
cDNA of clone W36 could be isolated successfully by 5’ RACE
PCR, whereas the recovery of the missing 5’ and 3’ ends of Z25
was only partially successful yielding a 630 bp cDNA fragment.
However, the Z25 translated polypeptide showed 93% identity
and 99% similarity with W36, suggesting that the two sequences
encode proteins with similar properties. Hence, only W36 was cho-
sen for further analysis and termed DluHKS. The full-length cDNA
sequence of DluHKS was 1442 bp containing a 133 bp 5’-non-cod-
ing region, a 1170 bp open reading frame (ORF) with start- and
stop-codons encoding a protein with 389 amino acid residues
and a 139 bp 3’-non-coding region. The predicted molecular
weight of the encoded protein is 42.8 kDa.
Fig. 4. SDS–PAGE analysis of the purification of recombinant DluHKS. The protein
from each individual purification step was applied to a 10% SDS gel and visualized
with Coomassie Brilliant Blue R250. Lanes: 1 molecular mass markers (kDa), 2 non-
induced E. coli cells, 3, 4 and 5 IPTG-induced cells after 2, 4 and 6 h at 24 °C, 6 total
cellular lysate, 7 flow-through of affinity column, 8 DluHKS purified by affinity
chromatography on cobalt resin. The position of recombinant DluHKS is indicated
by an arrow.
The nucleotide sequence of DluHKS will appear in the DDBJ/
EMBL/GenBank databases with the accession number EF405822.
The translated polypeptide sequence of DluHKS showed 51–67%
identity with plant type III PKSs and ca. 20% identity with bacterial
type III PKSs. Highest identities (69%) were found with a CHS of Vi-
tis vinifera, while the identity to P. indica HKS (PinHKS), which has
been proposed to be involved in naphthoquinone formation, was
only 58%. DluHKS contains the conserved Cys-His-Asn catalytic
triad-like other type III PKSs (Fig. 2). In addition, several residues
that are important for the active-site geometry and for the steric
modulation of enzyme activity are conserved between DluHKS
and CHS, e.g. Gly211, Phe215, Phe265 and Pro375 (numbering
according to Medicago sativa CHS2) (Ferrer et al., 1999; Austin
and Noel, 2003). However, in comparison with M. sativa CHS2,
the sterically important active site positions Ser133, Thr197,
Gly256 and Ser338 are changed to Ala, Phe, Leu and Thr, respec-
tively. Most of these exchanges are also found in the sequence of
PinHKS, except for the residue at position 197, which is Ala in Pin-
HKS (Fig. 2).
A phylogenetic analysis was performed with plant, bacterial,
fungal and slime mold type III PKSs using Escherichia coli (E. coli)
b-ketoacyl synthase III as outgroup (Fig. 3). In the tree, plant PKSs
form a distinct cluster, and CHSs and most stilbene synthases
(STSs) group separately from plant PKSs with other functions. De-
spite its higher sequence similarity to CHSs, DluHKS appears in the
non CHS cluster and seems to be most closely related to PinHKS
and the heptaketide forming aloesone synthase (ALS) of Rheum pal-
matum (Abe et al., 2004b). This suggests that DluHKS is function-
ally similar to the two other PKSs and may therefore be able to
perform five or six condensation steps with a short starter. How-
ever, conclusions drawn from alignments or cladistic analyses
can be misleading and need to be confirmed by functional
characterization.
Fig. 5. Radio-TLC analysis of products extracted from PKS assays. Comparison of
products obtained with recombinant CalCHS1 (1), PinHKS (2) and DluHKS (3) in
incubations with acetyl-CoA (5) and [2-14C]malonyl-CoA (1). The compounds ide-
ntified as triacetate lactone (TAL, 11) and 6-(2’, 4’-dihydroxy-6’-methylphenyl)-4-
hydroxy-2-pyrone (10) are marked by arrows.
mass of 46 kDa which includes the hexahistidine tag and a few
amino acids from the vector.
Consequently, DluHKS was expressed in E. coli. The ORF was
cloned into the pET-100/D-TOPO vector containing an N-terminal
hexahistidine fusion tag. The expression vector was transformed
in the host E. coli BL21 (DE3). After induction of expression with
IPTG, the recombinant protein was purified by immobilized metal
affinity chromatography on cobalt resin. SDS–PAGE results are
shown in Fig. 4. The purified enzyme gave a band with a molecular
Purified recombinant DluHKS was incubated with acetyl-CoA
(5) as starter and [2-14C]malonyl-CoA (1) as extender unit, and
the products extracted from the incubation were analyzed by
radio-TLC. For comparison, recombinant PinHKS and CHS1 from
Cassia alata (CalCHS1) were analyzed under the same conditions.
Radio-TLC analysis revealed that PinHKS and DluHKS are function-
ally similar and synthesized several compounds that comigrated

A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
3047
Table 1
Enzymatic products of DluHKS with different starter CoA esters

Table 2
Collision-induced dissociation (CID) mass spectra of enzymatically formed products obtained from the [MꢀH]ꢀ ions by LC/ESI-MS (RTLC = LC retention time) and elemental composition determined by HR/ESI-MS
Starter CoA
ester
Product
Collision
energy
RtLC
(min)
[MꢀH]-
Ions in the negative ion CID mass spectra [m/z (rel. int.)]
HR-negative ion
ESI-MS (elemental
composition)
(m/z)
Acetyl-CoA (5)
11
12
13
10
4.74
4.23
6.66
125
167
209
233
not determined
not determined
not determined
m/z 233.04516 [M-H]-
(calcd. for C12H9O5ꢀ
233.04555)
+15
+15
+15
167 (16), 125 (100), 123 (10), 83 (8), 81 (13), 75 (3), 65 (7)
209 (1), 125 (100), 123 (4), 81(2)
233 (24), 191 (14), 189 (100), 165 (41), 147 (54), 145 (3)
12.16
n-Butyryl-CoA
(14)
16
+15
8.52
195
195 (13), 151 (14), 125 (100), 123 (7), 109 (11) 83 (4), 65 (4)
m/z 195.06584 [M-H]-
(calcd. for C10H11O4ꢀ
195.06628)
15
17
+15
+15
11.33
16.92
153
237
153 (13), 111 (4), 110 (100)
237 (1), 151 (5), 125 (100), 123 (2), 81 (4)
m/z 237.07652 [M-H]-
(calcd. for C12H13O5ꢀ
237.07685)
Isovaleryl-CoA
(18)
20
19
21
+15
+20
+20
16.59
17.55
19.34
209
167
251
209 (19), 165 (13), 125 (100), 123 (14), 83 (3)
167 (1), 125 (11), 123 (100), 83 (5)
251 (3), 165 (2), 125 (100), 81 (2)
m/z 209.08142 [M-H]-
(calcd. for C11H13O4ꢀ
209.08194)
m/z 167.07108 [M-H]-
(calcd. for C9H11O3ꢀ
167.07137)
m/z 251.09215 [M-H]-
(calcd. for C13H15O5ꢀ
251.09250)
n-Hexanoyl-
CoA (22)
24
23
+15
+15
19.32
19.92
223
181
223 (22), 181 (1), 179 (22), 137 (8), 125 (100), 83 (3)
181 (14), 139 (5), 137 (100)
m/z 233.09714 [M-H]-
(calcd. for C12H15O4ꢀ
233.09758)
m/z 181.08657 [M-H]-
(calcd. for C10H13O3ꢀ
181.08718)
Benzoyl-CoA
(25)
27
26
+15
+15
16.53
17.75
229
187
229 (38), 187 (16), 185 (65), 161 (100), 145 (5), 143 (68), 119 (16), 83 (27), 65 (7)
187 (24), 145 (5), 143 (100)
m/z 229.05038 [M-H]-
(calcd. for C13H9O4ꢀ
229.05063)
m/z 187.03965 [M-H]-
(calcd. for C11H7O3ꢀ
187.04007)
Cinnamoyl-CoA
(28)
30
29
+15
+15
18.90
20.01
255
213
255 (73), 213 (6), 211 (38), 195 (7), 187 (100), 183 (4), 172 (18), 145 (36), 125 (6), 83 (3)
213 (30), 171 (4), 169 (100), 141 (8), 139 (2)
m/z 255.06693 [M-H]-
(calcd. For C15H11O4ꢀ
255.06628)
m/z 213.05525 [M-H]-
(calcd. for C13H9O3ꢀ
213.05572)
p-Coumaroyl-
CoA (2)
32
31
31
+15
+15
ꢀ20
14.74
16.35
271
229
231a
271 (64), 253 (7), 229 (37), 227 (42), 211 (56), 203 (75), 185 (13), 179 (8), 161 (19), 145 (100), 125 (40)
229 (37), 187 (5), 185 (100), 157 (3), 143 (26)
m/z 271.06114 [M-H]-
(calcd. for C15H11O5ꢀ
271.06120)
m/z 229.05031 [M-H]-
(calcd. for C13H9O4ꢀ
229.05063)
231 (25), 213 (12), 203 (23), 185 (27), 171 (19), 157 (38), 147 (100), 145 (6), 129 (5), 121 (11), 107 (4), 69 (9)b
[M+H]+.
Positive ion CIDMS.
a
b

A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
3049
(Fig. 5). In contrast, CalCHS1 produced mainly triacetate lactone
(TAL, compound 11, Table 1) from acetyl-CoA (5) and malonyl-
CoA (1).
onyl-CoA (1) into products was found with n-butyryl-CoA (14), iso-
valeryl-CoA (18) and n-hexanoyl-CoA (22). For comparison, a three
times lower incorporation of [2-14C]malonyl-CoA (1) was deter-
mined with acetyl-CoA (5) as primer unit. This result suggests that
acetyl-CoA (5) might not be the physiological substrate. However,
these assays with different start substrates were carried out under
conditions used by Samappito et al. (2002), which are not opti-
mized for the formation of the hexaketide (10). In addition, there
are reports on type III PKSs that show higher affinity to unnatural
substrates than to their physiological primer unit (Abe et al.,
2004a). Given the substrate promiscuity of type III PKSs, not only
the affinity for a certain substrate, but also its availability will
determine the physiological function of the enzyme.
The products synthesized from alternative starter CoA esters
could not be identified by radio-TLC analysis due to the lack of
authentic standards. Consequently, scaled up assays with unla-
beled substrates were performed, and the reaction products were
analyzed by LC/ESI-MS/MS. High resolution (HR)/ESI-MS analysis
was carried out to determine the elemental composition of the
products. The chemical structures of the products are summarized
in Table 1 and the corresponding mass-spectral data are shown in
Table 2. Recombinant DluHKS produced tri-, tetra- and pentaketide
pyrones with the small primer units n-butyryl-CoA (14) and iso-
valeryl-CoA (18). With the larger starter CoA esters hexanoyl-CoA
(22), benzoyl-CoA (25), cinnamoyl-CoA (28) and p-coumaroyl-
CoA (2) only di- and triketide pyrones were formed. Bis-noryango-
nin (31), the triketide pyrone from the starter p-coumaroyl-CoA
(2), was identified by its parent ions [M+H]+ (m/z 231) and [MꢀH]ꢀ
(m/z 229), respectively (Table 2). The electrospray CID spectrum of
the [MꢀH]ꢀ ion (m/z 229) displayed key ions at m/z 187
[MꢀHꢀCH2CO] and m/z 185 [MꢀHꢀCO2], and the CID spectrum
of the [M+H]+ ion (m/z 231) yielded a characteristic fragment at
m/z 147 (Samappito et al., 2002).
The major product of DluHKS is the hexaketide 6-(2’,4’-dihy-
droxy-6’-methylphenyl)-4-hydroxy-2-pyrone (compound 10, Ta-
ble 1). This compound was identified on the radio-TLC plate by
co-chromatography with an authentic standard prepared by
hydrolysis of aloenin A. With the exception of TAL (11), which
could be tentatively identified by co-migration with the main
product of CalCHS1, and the hexaketide 10, the identity of all other
radiolabeled products of DluHKS in the TLC analysis could not be
determined because authentic standards were not available.
If the assay mixture was not stopped by acidification and di-
rectly spotted onto the TLC plate, most of the radioactive products
remained at the origin or close to it (data not shown). This suggests
that several products are only formed after acidification. Most
likely, the recombinant PinHKS and DluHKS produce linear polyke-
tides that undergo cyclization at low pH.
To further identify the products formed, DluHKS was incubated
with unlabeled acetyl-CoA (5) and malonyl-CoA (1) in scaled up as-
says. The products were analyzed by HPLC, LC/ESI-MS and GC/MS
(HPLC and GC/MS data not shown). By comparison with the CID
(collision induced dissociation) mass spectra of the products of
PinHKS (Springob et al., 2007), the polyketides synthesized by
DluHKS were identified as 4-hydroxy-6-methyl-2-pyrone (TAL,
11) and 6-acetonyl-4-hydroxy-2-pyrone (12), 4-hydroxy-6-(2’,4’-
dioxo-pentyl)-pyrone (13) and 6-(2’,4’-dihydroxy-6’-methyl-
phenyl)-4-hydroxy-2-pyrone (10) (Tables 1 and 2). However, one
product of PinHKS, 4-hydroxy-6-(2’,4’-trioxo-heptyl)-pyrone, was
not found to be synthesized by DluHKS.
Recombinant DluHKS does not depend on acetyl-CoA (5) as
start substrate, because it can also use malonyl-CoA (1) efficiently.
However, in comparison to assays without acetyl-CoA (5), 22%
higher yields (p = 0.057) of the hexaketide phenylpyrone (com-
pound 10) were obtained when acetyl-CoA (5) was added to the as-
say mixture. Even in the presence of 100-fold higher
concentrations of unlabeled malonyl-CoA (1), [1-14C]acetyl-CoA
(5) was utilized as starter substrate.
Acylphloroglucinol derivatives or stilbenes, the typical products
of CHSs and STSs, could not be detected with any of the starters.
Hence, the product spectrum of DluHKS is similar to that obtained
with PinHKS (Springob et al., 2007).
It is known from previous investigations that the active site cav-
ity of type III PKSs acts as size-based filter and determines starter
substrate specificity, number of possible extensions and type of
cyclization. Therefore, changes in amino acid residues lining the
active site can lead to enzymes with altered product profiles (Jez
et al., 2000a; Abe et al., 2006a,b). In order to find out whether mod-
ifications in the active site can explain the unusual properties of
DluHKS and PinHKS, the enzymes were modeled based on the
structure of M. sativa CHS2 complexed with resveratrol (PDB code
1CGZ) using the SWISS-MODEL protein modeling server.
Although the overall three-dimensional fold of DluHKS and Pin-
HKS is very similar to that of CHS, several amino acids lining the
active site cavity of the HKSs are changed relative to M. sativa
CHS2 (Fig. 6). For example, Ser133, Gly256 and Ser338 (numbering
according to M. sativa CHS2), which are highly conserved in CHSs,
are substituted with Ala, Leu and Thr, respectively, in the two
HKSs. In addition, Thr194, Val196 and Thr197, three amino acids
at the bottom of the active site of CHS, are replaced by Phe202,
Phe204 and Ala205 in PinHKS. DluHKS, instead, contains Thr192,
Ile194 and Phe195 at the respective positions. Although the two
HKSs show a different amino acid substitution pattern in this case,
most substitutions introduce bulkier residues suggesting that the
available space may be reduced in comparison with CHS. Several
type III PKSs functionally divergent from CHS contain similar ami-
no acid exchanges. In particular, mutations of T197, G256, and
S338 were shown to modulate PKS activity. Analysis of the three-
dimensional structures of Gerbera hybrida 2-pyrone synthase
(2-PS) and A. arborescens PCS demonstrated that the small to large
exchange of amino acids corresponding to T197, G256 and S338 in
Since the hexaketide (compound 10) is the main product of re-
combinant DluHKS with acetyl-CoA (5) as starter substrate, the ki-
netic parameters of its formation were investigated. Formation of
10 is linear during a period of 20 min using protein concentrations
up to 2 lg/100 ll. DluHKS showed a pH optimum for the formation
of 10 at pH 6.0 and 6.5 and a temperature optimum at 45 °C. KM
values of 31 lM for acetyl-CoA (5) and 83 lM for malonyl-CoA
(1) were found. These values are about 10–20-fold higher than
the KM values determined for the starter and extender substrates
(p-coumaroyl-CoA, 2, and malonyl-CoA, 1) of CHSs and STSs (Schüz
et al., 1983; Schöppner and Kindl, 1984; Jez et al., 2000b). How-
ever, comparatively high KM values for malonyl-CoA (1) were also
reported for Aloe arborescens pentaketide chromone synthase (PCS)
and octaketide synthase, two PKSs that catalyze more condensa-
tion steps than most other type III PKSs (Abe et al., 2005a,b).
To determine the activity of DluHKS with other starter mole-
cules, the recombinant enzyme was incubated with [2-14C]malo-
nyl-CoA (1) and the CoA esters acetyl-CoA (5), n-butyryl-CoA
(14), isovaleryl-CoA (18), n-hexanoyl-CoA (22), benzoyl-CoA (25),
cinnamoyl-CoA (28) and p-coumaroyl-CoA (2). The resulting radio-
active products were resolved by reversed-phase TLC (data not
shown). DluHKS showed relaxed starter unit specificity, accepted
both aliphatic and aromatic CoA esters as primers for polyketide
synthesis and produced multiple products from each. To compare
the amount of products formed from acetyl-CoA with the other
starter CoA esters, the total incorporation of radioactivity from la-
beled malonyl-CoA (1) was used for quantification. Under the
experimental conditions used, highest incorporation of [2-14C]mal-

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A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
Fig. 6. Models of the active sites of M. sativa CHS2, DluHKS and PinHKS. The structures of DluHKS and PinHKS were modeled based on the structure of M. sativa CHS2
complexed with resveratrol (PDB code 1CGZ, resveratrol not shown).
CHS reduces the volume of the active site cavity (Jez et al., 2000a;
Morita et al., 2007). Similarly, Streptomyces coelicolor 1,3,6,8-tetra-
hydroxynaphthalene synthase (THNS) contains Tyr instead of
G256, which leads to a horizontal restriction of the cavity and a
preference of small starter molecules. T197 and S338, however,
are replaced by smaller amino acids in THNS. This causes a down-
ward expansion that allows four extensions with malonyl-CoA (1)
(Austin et al., 2004).
The reduced space available in the active site can explain the
preference of DluHKS for smaller CoA esters. However, it is still un-
clear why the HKSs can catalyze more condensations with malo-
nyl-CoA (1) than CHS. Most likely, other amino acid exchanges,
for example the S133A substitution, which occurs in DluHKS and
PinHKS, might increase the space available for the growing polyke-
tide chain in the active site of DluHKS. The importance of the ami-
no acid residue in position 133 and its neighbors was shown with
Ruta graveolens acridone synthase 2 (ACS2). Site-directed mutagen-
esis of S132, A133 and V265 into Thr, Ser and Phe, the correspond-
ing residues of M. sativa CHS2, converted ACS2 into a functional
the biosynthesis of stilbenecarboxylates and anthrones, the pres-
ence of such an enzyme has been hypothesized (Eckermann
et al., 2003; Abe et al., 2005b). In the case of naphthoquinone bio-
synthesis, additional evidence for the presence of a reductase can
be derived from biomimetic syntheses of acetate-derived naphtha-
lenes and isoquinolines (Bringmann, 1985). A chemically prepared
2,4,6,8,10-pentaketone spontaneously undergoes aldol cyclization
between C-2 and C-7, yielding a methylresorcinol ring identical
to that of the hexaketide 10. In contrast, if the oxygen at C-6 of
the pentaketone is protected, e.g., as acetal, the condensation oc-
curs between C-4 and C-9, a cyclization identical to the postulated
first ring closure in naphthoquinone biosynthesis (Fig. 1, number-
ing of carbon atoms according to polyketide literature). Interest-
ingly, the oxygen at C-7 of the hexaketide intermediate (Fig. 1),
corresponding to C-6 of the pentaketone, is removed by reduction
in all naphthoquinones of polyketide origin. Although the cited
cyclization experiments were performed with a pentaketone and
not with the herein postulated hexaketide intermediate, the chem-
istry of the cyclization reaction is valid for both molecules. For the
biosynthesis of naphthoquinones this suggests that the cyclization
of the first ring between C-4 and C-9 occurs after the oxygen at C-7
has been reduced.
The postulation of enzymes acting downstream of DluHKS
in vivo also implies shuttling of a hexaketide intermediate and pos-
sibly interactions with the other proteins. The active site of type III
PKSs is buried in the enzyme, but it is only separated from the out-
side solution by a b-strand finger. Interactions with other proteins
may displace this thin wall and thus reshape the active site cavity
(Austin and Noel, 2003). The suggestion of accessory proteins for
naphthoquinone biosynthesis remains highly speculative, but the
EST library of D. lusitanicum callus cultures may provide a good
base to identify and study such additional co-factors.
ˇ
CHS (Lukacin et al., 2001).
Two other interesting amino acid exchanges relative to CHS oc-
cur only in PinHKS, but not in DluHKS. Gly211 and Phe265 are re-
placed with Cys and Ile, respectively. These variations do not
influence the ability to synthesize hexaketides from six acetate
units, however, they might be responsible for the slightly different
product pattern of the two enzymes.
This study had the aim to isolate a PKS from D. lusitanicum that
catalyzes the formation of naphthalenes or naphthoquinones.
However, in analogy to the previously described PinHKS, the re-
combinant DluHKS could not catalyze the formation of naphtha-
lenes, but produced a-pyrones from an acetyl-CoA (5) starter and
two to five malonyl-CoA (1) extender units. To the best of our
knowledge, D. lusitanicum does not produce any of the pyrone-type
compounds detected (Table 1). On the other hand, we could not
observe naphthalene formation in in vitro PKS assays with crude
protein extracts from D. lusitanicum callus cultures (data not
shown). Nevertheless, DluHKS performs five condensations as re-
quired for the synthesis of the naphthoquinone skeleton. There-
fore, it appears possible that DluHKS provides the backbone of
these metabolites, and further downstream processing steps con-
vert the intermediate into naphthoquinones.
It is still possible that DluHKS is involved in a different biosyn-
thetic pathway. Moreover, the recombinant enzyme may produce
naphthalenes under different conditions than applied in the
in vitro assays. Since the enzyme described is the second HKS iso-
lated from a naphthoquinone-producing plant, however, it be-
comes more likely that this kind of PKS provides the backbone of
naphthoquinones.
3. Conclusion
Most likely, a reductase is required that removes the oxygen at
the third acetate unit. The only polyketide reductase characterized
from plants so far is chalcone reductase. For other pathways, e.g.
We have isolated a PKS that synthesizes a-pyrones from three
to six acetate units. The enzyme shows the promiscuous substrate

A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
3051
acceptance typical of most known type III PKS, although a clear-cut
answer about its physiological function could not be provided.
However, our results suggest that enzymes like DluHKS and Pin-
HKS that can condense six acetate units catalyze the first step of
naphthoquinone biosynthesis.
exclusion chromatography, sequencing reactions were run on the
ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems).
The EST sequences were trimmed for vector contamination and
assembled with the SeqManIITM module of the Lasergene software
package (DNASTAR Inc., Madison, WI, USA) to obtain individual
contigs. The assembling parameters included a minimum match
size of 20 and a minimum match percentage of 80. Each contig
was subjected to an automated BlastX search using DNASISTM (Hit-
achi Software Engineering, Berlin Germany).
4. Experimental
4.1. Chemicals
4.5. 5’ RACE PCR for full-length cDNA synthesis
Acetyl-CoA, malonyl-CoA (1), n-butyryl-CoA (14), isovaleryl-
CoA (18), n-hexanoyl-CoA (22), benzoyl-CoA (25), and plumbagin
(8) were purchased from Sigma (Munich, Germany). [1-14C]acet-
yl-CoA (5) (4 mCi/mmol) and [2-14C]malonyl-CoA (1) (55 mCi/
mmol) were obtained from Biotrend Chemikalien (Cologne, Ger-
many). p-Coumaroyl-CoA (2) and cinnamoyl-CoA (28) were kindly
provided by Dagmar Knöfel at the Leibniz-Institut für Pflanzenbi-
ochemie (IPB), Halle (Saale), Germany. Aloenin A was purchased
from Phytoplan GmbH (Heidelberg, Germany).
The missing 5’ end of the DluHKS cDNA was isolated by 5’-rapid
amplification of cDNA ends (5’ RACE) using the BD SMARTTM RACE
cDNA Amplification Kit (Clontech, Heidelberg, Germany). Total
RNA of D. lusitanicum callus cultures was used as template for
first-strand cDNA synthesis. 5’ RACE PCR was performed using
the gene-specific primer 5’-GTA CTC GCT GAG CAC GTA TCG ACT
CTCC-3’ and the universal primer mix (UPM) supplied by the man-
ufacturer to amplify an approximately 1.2 kb DNA fragment. Cy-
cling conditions consisted of 5 cycles of 94 °C, 30 s; 68 °C; 30 s
and 72 °C, 3 min, followed by 5 cycles of 94 °C, 30 s; 66 °C, 30 s
and 72 °C, 3 min, followed by 5 cycles of 94 °C, 30 s; 64 °C, 30 s
and 72 °C, 3 min, followed by 25 cycles of 94 °C, 30 s; 62 °C, 30 s
and 72 °C, 3 min and followed by 5 min at 72 °C. The 5’ RACE PCR
product was purified by gel electrophoresis and extracted using
the Mini Elute Gel Extraction Kit (Qiagen), ligated into pGEMÒ T-
Easy (Promega, Madison, WI, USA) and sequenced.
4.2. Plant material
Callus cultures of D. lusitanicum were grown on LS medium sup-
plemented with 0.186 mg/l a-naphtylacetic acid and 0.22 mg/l 2,4-
dichlorphenoxy acetic acid. The callus was subcultured every 4
weeks and maintained under a 16 h photoperiod at 24 °C.
4.3. cDNA library construction
4.6. Functional expression in E. coli
Total RNA was isolated from callus cultures of D. lusitanicum as
described by Salzman et al. (1999). Poly (A)+ RNA was obtained
from total RNA using the OligotexTM mRNA Kit (Qiagen, Hilden,
Germany), and synthesis of first- and second-strand cDNA was car-
ried out according to the instructions of the CloneMinerTM cDNA Li-
brary Construction Kit (Invitrogen, Karlsruhe, Germany). The
double strand cDNA was ligated with the attB adapter and size
fractionated to remove excess primer, adapters, and small cDNAs
by CC (SephacrylÒ S-500 HR resin). The attB-flanked cDNA was li-
gated into pDONRTM222 (attP containing vector, Invitrogen) with
BP ClonaseTM enzyme mix through the GatewayÒ BP recombina-
tion reaction and transformed into competent Escherichia coli
ElectroMaxTM DH10BTM T1 Phage Resistant Cells (Invitrogen). The
non-amplified D. lusitanicum cDNA library contained 5 ꢁ 106
colony forming units in a total volume of 12 ml.
To express the DluHKS full-length cDNA in E. coli, the open read-
ing frame was cloned into pET-100D/TOPO (Invitrogen), which
contains a hexahistidine N-terminal fusion tag. Amplification was
performed using the forward primer: 5’-CAC CAT GGC ATT TGT
GGA GGG AAT GGG GAAG-3’ (the 4 underlined nucleotides anneal
to the GTGG overhang of the pET100/D-TOPO vector) and the
reverse primer: 5’-TTA GTT GTT TAT AGG GAA GCT TCG CAA
TAC-3’ with D. lusitanicum first-strand cDNA as template and Pfu
DNA polymerase (Fermentas, St.Leon-Rot, Germany). The 1.2 kb
PCR product was gel-purified and ligated into pET-100D/TOPO.
The sequence of the resulting construct DluHKS:pET-100D/TOPO
was confirmed, and the expression plasmid was transformed into
E. coli BL21 StarTM (DE3) One ShotÒ cells (Invitrogen).
E. coli harboring DluHKS:pET-100D/TOPO were grown at 37 °C
in LB medium containing 50 lg/ml ampicillin until A600 ꢂ 0.6. Pro-
tein expression was induced with 1.0 mM isopropyl-1-thio-b-D-
4.4. Expressed sequence tag (EST) analysis of cDNA library
galactopyranoside, and the culture was grown at 24 °C for 6 h.
E. coli cells were harvested, and recombinant DluHKS was purified
by following procedures described previously (Samappito et al.,
2002, 2003).
Hundred microlitre of a 1:100 dilution of the D. lusitanicum
cDNA library was plated onto prewarmed LB plates containing
50 lg/ml kamamycin and incubated overnight at 37 °C. Single col-
onies were picked and grown in 50 ll Luria–Bertani (LB) containing
kamamycin at 37 °C overnight. cDNA inserts were amplified from
the bacterial cultures by PCR using an M13 uni primer: 5’-ACG
ACG TTG TAA AAC GAC GGC CAG-3’ and an M13 reverse primer:
5’-TTC ACA CAG GAA ACA GCT ATG ACC-3’. The PCR reaction was
performed in 50 ll total volume containing 1–2 ll of the bacterial
culture, 5 ll of 10ꢁ PCR buffer with 15 mM MgCl2, 1 ll dNTPmix,
1 ll of 10 lM M13 uni primer and 1 ll of 10 lM M13 reverse pri-
mer under the following PCR conditions: 5 min at 94 °C for 1 cycle,
then 30 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s. This
was followed by an additional elongation step at 72 °C for 5 min.
The PCR products were sequenced using the M13 uni primer and
the BigDyeÒ Terminator v3.1 Cycle sequencing Kit (Applied Biosys-
tems, Darmstadt, Germany). After removal of excess dye by size
4.7. Polyketide synthase assay
The standard reaction mixture contained 100 mM K-Pi buffer
(pH 6.5), 50 lM acetyl-CoA (5), 100 lM [2-14C]malonyl-CoA (1)
(70,000 dpm) and 0.5–5.0 lg recombinant enzyme in a 100 ll reac-
tion. The assays were incubated for 15 min at 45 °C, stopped by
addition of 10 ll of 1 M HCl, incubated for an additional 15 min
at 45 °C to convert all uncyclized products into lactones, and ex-
tracted with EtOAc (3 ꢁ 500 ll). The organic phases were dried,
and products were separated by TLC on silica gel with EtOAc–
MeOH–H2O (100:16.5:13.5; v/v). The 14C-labeled products were
visualized by phosphorimagery (Typhoon 9410, Amersham Biosci-
ences). TAL (11) was identified by comparison with the products of
chalcone synthase from Cassia alata (CalCHS1) (Samappito et al.,

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A. Jindaprasert et al. / Phytochemistry 69 (2008) 3043–3053
2002), incubated under the same conditions as DluHKS, and 6-
(2’,4’-dihydroxy-6’-methylphenyl)-4-hydroxy-2-pyrone (10) was
identified as described in Section 4.10.
H2O–CH3CN (98:2; v/v) each with 0.2% HOAc, with the following
gradient: from 0% B to 60% B in 25 min, 25–30 min 60% B, 30–
32 min 60–100% B, 32–35 min 100% B at a flow rate of 1 ml minꢀ1
The detection wavelength was set to 300 nm.
.
The kinetic parameters of DluHKS were determined by
quantifying the spot of the hexaketide (10) on the TLC by phos-
phorimaging. All assays were carried out in triplicate. The KM for
malonyl-CoA (1) was determined using 50 lM acetyl-CoA (5) and
5–200 lM [2-14C]malonyl-CoA (1). To determine the KM of
acetyl-CoA (5), the incubations contained 3–81 lM [1-14C]acetyl-
CoA (5) and 500 lM unlabeled malonyl-CoA (1). As standard for
quantification, 1 ll of 1 mM [2-14C]malonyl-CoA (1) (7000 dpm/
ll) was spotted on each TLC plate. For the KM of acetyl-CoA (5),
1 ll of 1.6 mM [1-14C]acetyl-CoA (5) (14,000 dpm/ll) and 1 ll of
0.17 mM [1-14C]acetyl-CoA (5) (1460 dpm/ll) were spotted on
the TLC plates as standards.
To compare the activity of DluHKS with acetyl-CoA (5) and var-
ious larger starter CoA esters, the incubations were performed as
described by Samappito et al., 2002. For quantitative evaluation
of these assays, the incorporation of [2-14C]malonyl-CoA (1) into
all reaction products from one starter substrate was quantified
by phosphorimaging. The enzyme assay contained 100 mM HEPES
buffer (pH 7.0), 20 lM starter CoA, 70 lM [2-14C]malonyl-CoA (1)
(80,000 dpm), and 5 lg polyketide synthase in a 50 ll reaction vol-
ume. The assay mixture was incubated at 30 °C for 30 min. The
reaction was stopped by the addition of 5 ll 10% (v/v) HCl and
was extracted with EtOAc (2 ꢁ 100 ll). The combined organic
phases were evaporated to dryness and products separated by
RP-18 thin layer chromatography (Merck) and developed in
MeOH–H2O–HOAc (75:25:1; v/v). Each assay was carried out in
duplicate.
4.10. Preparation of 6-(2’,4’-dihydroxy-6’-methylphenyl)-4-hydroxy-
2-pyrone (10)
Compound 10 was obtained by hydrolysis of aloenin A (6-(2’-O-
b-D-glucopyranosyl-4’-hydroxy-6’-methylphenyl)-4-methoxy-
2-pyrone) in MeOH–H2O–HCl (80:17:3; v/v) as described
previously (Springob et al., 2007). Although this yielded
mainly 6-(2’,4’-dihydroxy-6’-methylphenyl)-4-methoxy-2-pyrone,
10 was obtained as minor by-product and could be purified by
HPLC (Rt = 15.9 min) using the conditions described under Section
4.9. The identity of 10 was confirmed by LC/ESI-MS.
Acknowledgements
We thank Verona Dietl, Christine Kuhnt and Dagmar Knöfel
(Leibniz-Institut für Pflanzenbiochemie, Halle, Germany) for tech-
nical assistance and Dr. Joseph M. Jez (Donald Danforth Plant Sci-
ence Center, St. Louis, MO, USA) for his help in preparing the
models of DluHKS and PinHKS. A. Jindaprasert was supported by
the Royal Golden Jubilee Ph.D. program, Thailand Research Fund
(RGJ-TRF), the German Academic Exchange Service (DAAD), and
Thailand’s National Center for Genetic Engineering and Biotechnol-
ogy (BIOTEC, Grant No. 2/2545). Financial support by the DFG pri-
ority program SPP1152: ‘‘Evolution of metabolic diversity”
(KU990/5-2) is gratefully acknowledged.
4.8. LC–MS and high resolution MS analysis of enzymatic products
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To identify the enzymatic products in the incubation mixtures
containing various starter-CoA esters, 5 scaled-up 200 ll reactions
were carried out containing 100 mM HEPES (pH 7.0), 50 lM star-
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After incubation for 1 h at 30 °C, the mixtures were acidified and
extracted with EtOAc (2 ꢁ 400 ll). The combined organic phases
were evaporated to dryness, and the residue was dissolved in
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