An unusual thioesterase promotes isochromanone ring formation in ajudazol biosynthesis

Article abstract of DOI:10.1002/cbic.200900712

The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) multienzyme "assembly line" in the myxobacterium Chondromyces crocatus Cm c5. The most striking structural feature of

Full text of DOI:10.1002/cbic.200900712

DOI: 10.1002/cbic.200900712
An Unusual Thioesterase Promotes Isochromanone Ring
Formation in Ajudazol Biosynthesis
[
a]
Kathrin Buntin, Kira J. Weissman, and Rolf Mꢀller*
The ajudazols are antifungal secondary metabolites produced
by a hybrid polyketide synthase (PKS)-nonribosomal peptide
synthetase (NRPS) multienzyme “assembly line” in the myxo-
bacterium Chondromyces crocatus Cm c5. The most striking
structural feature of these compounds is an isochromanone
ring system; such an aromatic moiety is only known from two
other complex polyketides, the electron transport inhibitor
stigmatellin and the polyether lasalocid. The cyclization and
aromatization reactions in the stigmatellin pathway are pre-
sumed to be catalyzed by a cyclase domain located at the end
of the PKS, while the origin of the lasalocid benzenoid ring
remains obscure. Notably, the ajudazol biosynthetic machinery
does not incorporate a terminal cyclase, but instead a variant
thioesterase (TE) domain. Here we present detailed phyloge-
netic and sequence analysis, coupled with experiments both in
vitro and in vivo, that suggest that this TE promotes formation
of the isochromanone ring, a novel reaction for this type of
domain. As the ajudazol TE has homologues in several other
secondary-metabolite pathways, these results are likely to be
generalizable.
Introduction
Over the last several decades, myoxbacteria have increasingly
gained recognition as a source of bioactive natural com-
nal nucleophile (e.g., ꢀOH or ꢀNH ) on the product chain,
2
thereby releasing a macrocyclic compound. Macrolactone
products are generated, for example, during biosynthesis of
the polyketide erythromycin A, and the nonribosomal peptide
[
1,2]
pounds.
ketide synthases (PKS), nonribosomal peptide synthetases
NRPS) and hybrids of the two systems. PKS and NRPS are
Most of these metabolites are produced by poly-
[
9,10]
(
surfactin,
while the mixed polyketide–nonribosomal pep-
[11]
large, multimodular enzymes that are responsible for the gen-
eration of natural products by stepwise assembly from simple
tide metabolite vicenistatin is released as a macrolactam.
Numerous PKS and NRPS biosynthetic gene clusters also in-
corporate a second type of TE domain (a “type II TE” (TEII)) that
is not fused to the multienzyme assembly line. As inactivation
of TEII genes was typically found to reduce but not abolish
[3–6]
acyl-coenzyme A thioesters and amino acids, respectively.
Each module consists of multiple specialized catalytic domains,
which together exhibit all the enzymatic functions required for
the activation and incorporation of a single building block into
the growing chain, as well as its optional modification. The
common catalytic strategy used by PKS and NRPS is based
around carrier protein (CP) domains (acyl carrier protein (ACP)
and peptidyl carrier protein (PCP), respectively), which are
present in each module. The intermediates are tethered as
thioesters to the 4’-phosphopantetheine (Ppant) co-factor of
these CP domains, a covalent linkage that facilitates the direct-
ed transport of the growing chains through the biosynthetic
systems. Addition of the prosthetic group from coenzyme A is
carried out by a dedicated enzyme called a phosphopantethei-
nyltransferase (PPTase).
[12–16]
metabolite yields,
TEIIs were postulated to play a role in
maintaining functional assembly lines by regenerating blocked
systems. In PKS, such blockages can arise from aberrant decar-
boxylation of chain-extender units in the absence of an accept-
or substrate; this reaction results in ACP domains charged with
short, fatty acyl thioesters such as acetate and propionate. Re-
moval of the decarboxylation product by the TEII followed by
hydrolysis would liberate the CP, allowing it to continue its
normal operations. Consistent with this hypothesis, studies of
a number of PKS TEIIs in vitro have revealed a preference for
hydrolysis of acetate, propionate and butyrate over their car-
[17–20]
boxylated equivalents or longer acyl chains.
Evidence ob-
Chain extension continues until the intermediates reach the
end of the assembly line, whereupon they are typically transa-
cylated from the Ppant arm of the final carrier protein onto
the active-site serine of a terminal thioesterase (TE) domain.
This “type I TE” then catalyzes the controlled off-loading of the
product. One mode of chain release is hydrolysis, in which the
final intermediate is transferred to water as an external nucleo-
phile. This detachment strategy is used, for example, during
the biosynthesis of the myxobacterial metabolites spirangien
tained in vitro also supports a role for NRPS TEIIs in the release
of nonproductive acyl thioesters from the corresponding
[
a] K. Buntin, Dr. K. J. Weissman, Prof. Dr. R. Mꢀller
Helmholtz Institute for Pharmaceutical Research
Helmholtz Center for Infection Research and
Department of Pharmaceutical Biotechnology
P.O. Box 151150, 66041 Saarbrꢀcken (Germany)
Fax: (+49)681-30270202
E-mail: rom@mx.uni-saarland.de
[
7,8]
and DKxanthene.
Alternatively, and more commonly, the TE
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900712.
catalyzes intramolecular nucleophilic attack of a suitable inter-
ChemBioChem 2010, 11, 1137 – 1146
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R. Mꢀller et al.
[21]
PCPs. Such modifications
could occur during post-
translational modification
of the multienzymes, in
which an acyl-phosphopan-
tetheine (e.g., from acetyl-
CoA) was added to the PCP
by the PPTase instead of
the unmodified phospho-
pantetheine (from CoA). In
principle, this type of error
could also take place
during activation of PKS
polypeptides.
In addition, TEIIs from
NRPS pathways have been
shown to exhibit hydrolytic
activity towards a range of
amino acids; this observa-
tion suggests that they
might also repair errors in
[21,22]
substrate loading.
Al-
though it remains to be
demonstrated directly, TEII
genes from mixed PKS-
NRPS clusters would also
be expected to have both
activities. In general, the
overall broad specificity of
the type II TE hydrolase
family is consistent with a
model for TEII function in
which the enzymes act
slowly and fairly indiscrim-
inately to off-load stalled
acyl groups from CP do-
[17]
mains.
The gene cluster respon-
sible for the biosynthesis of
the antifungal ajudazols A
[23,24]
(
1) and B (2)
in the
myxobacterium Chondro-
myces crocatus Cm c5 con-
tains a single TE domain
that is located at the end
of the final subunit, AjuH,
in the hybrid PKS/NRPS as-
sembly line (type I organi-
zation; Figure 1). This find-
ing is unremarkable, except
that the ajudazols incorpo-
rate an isochromanone ring
that, on the basis of bio-
synthetic logic, should be
generated during off-load-
ing of the chain from the
terminal module. In the
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ChemBioChem 2010, 11, 1137 – 1146

Promotion of Isochromanone Ring Formation
pathway to stigmatellin (Scheme 1), one of two type I PKS sys-
tems known to generate aromatic moieties, cyclization and
aromatization to yield the final chromone ring appear to be
rating all type II TEs. Within these clades, the sequences form
subclades that generally correspond to their system of origin
(e.g., PKS, NRPS, or mixed PKS-NRPS). AjuTE and its closest
homologue, the type I TE from the jerangolid PKS gene cluster
[29]
in the myxobacterium Sorangium cellulosum (40% sequence
identity), clearly belong to the type II TE clade, and appear to
define a subclade together with a putative type I TE identified
in the genome sequence of the cyanobacterium Gloeobacter
violaceus PCC 7421.
Multiple sequence alignment of type I and II TEs shows that
both domain types incorporate the hydrolase signature se-
quence (Gly-Xaa-Ser-Xaa-Gly; residues 72–76, AjuTE number-
ing; Figure S1 in the Supporting Information). In the case of
type II TEs, the residue at position 75 in this motif is most
often a methionine. The sequence of AjuTE matches with
type II TEs in the signature region, including the M75, as does
JerTE. Furthermore, both TEs contain the highly conserved as-
partate (Asp179 in AjuTE) and histidine (His207), which togeth-
er with the catalytic serine (Ser74) form a catalytic triad in
[30,31]
type I and II TEs.
Nonetheless, the conserved His73 within
Scheme 1. Structures of complex polyketide metabolites incorporating un-
usual aromatic rings.
the core sequence of type II TEs is replaced in AjuTE and JerTE
by Ser and Cys, respectively. In the crystal structure of the
type II TE RifR, this His forms a hydrogen bond with the back-
bone carbonyl of the catalytic His, stabilizing its alignment
[31]
carried out by a novel cyclization domain that occupies the
within the triad. Consistent with a role in catalysis, mutation
of this residues (to an Ala) in the type II TE of the surfactin
pathway caused an approximately sevenfold reduction in turn-
[
25]
normal position of the TE. The mode of formation of the la-
salocid benzenoid ring (Scheme 1) remains unknown, although
[
26]
[32]
the PKS incorporates a typical type I TE domain. Together,
these observations raised the question of the role of the ajuda-
zol TE (AjuTE) in both chain release and isochromanone ring
formation. This uncertainty was reinforced by preliminary se-
quence analysis, which revealed that the closest homologues
to the AjuTE are type II proof-reading enzymes from PKS and
NRPS systems, not type I TEs as anticipated from its location in
over rate. Taken together, these observations suggested that
AjuTE and JerTE might behave like type II repair enzymes, but
equally, that the two domains might be inactive.
Expression, purification and kinetic analysis of a panel of TE
domains
[
27]
the multienzyme. We therefore aimed to obtain direct bio-
chemical evidence for the role of the terminal TE in ajudazol
biosynthesis. Using a combination of sequence analysis, enzy-
matic assays in vitro and gene inactivation, we provide evi-
dence for a new function of a TE domain in promoting the for-
mation of an aromatic ring.
In order to probe the activity and specificity of AjuTE directly,
we assayed the domain alongside representative type I and II
TE domains. We chose JerTE, a hydrolytic type I TE from the
spirangien PKS (SpiTEI), a macrocycle-forming TE from the
erythromycin A pathway (DEBSTEI), a bis-lactone-forming TE
from the hybrid PKS-NRPS responsible for disorazol biosynthe-
sis (DisTEI), and a type II TE from the tylosin polyketide gene
cluster (TylTEII; the structures of the pathway products are
shown in Figure S2). The expression constructs for the “type I”
TEs (Aju, Jer, Spi, and Dis) were designed to incorporate the
majority of the linker between the TE and the upstream ACP
domain (starting five amino acids C-terminal to the ACP), a
strategy that had already proved successful for the expression
Results
In silico analysis of TE domains
To evaluate the role of AjuTE in ajudazol biosynthesis, we ex-
[27]
tended our initial analysis of thioesterase sequences.
For
[33]
this, we created a phylogenetic tree of TE domains by using
of DEBSTEI. The genes were cloned into vector pET28b+ in
[
28]
Bayesian estimation (Geneious Pro 4.7.6) (Figure 2); impor-
tantly, generation of a phylogenetic tree by using the alterna-
tive neighbor-joining method yielded an equivalent result
order to obtain N-terminally His -tagged proteins, and expres-
6
sion was carried out in E. coli Rosetta BL21(DE3)pLysS/RARE
cells. DEBSTEI and TylTEII were produced according to the pub-
[17,33]
(data not shown). This analysis included 42 protein sequences
lished protocols.
All six recombinant proteins were then
representing all known types of TE domains (type I hydrolytic
and macrocyclization catalysts from PKS, NRPS and mixed PKS/
NRPS, as well as type II TEs from all three systems). Within the
tree, the TEs cluster into two distinct clades—one containing
all type I TEs, including the lasalocid TE, and a second incorpo-
purified to homogeneity by affinity chromatography (Figure 3),
and the identities of the newly engineered AjuTE, JerTE, DisTE,
and SpiTEI were confirmed by MALDI-MS analysis.
The substrate specificities of both type I and II TE domains
have been interrogated in vitro by using a range of carboxylic
ChemBioChem 2010, 11, 1137 – 1146
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1139

R. Mꢀller et al.
Figure 2. Phylogenetic tree analysis of various type I and II TE proteins from PKS and NRPS systems generated by using Bayesian estimation. The designations
TEI and TEII reflect whether the thioesterases exist as integral domains within a PKS or NRPS subunit (TEI) or as discrete proteins (TEII). The type of cluster to
which each TE belongs is given (P=PKS, N=NRPS, PN=PKS/NRPS hybrid), with the exception of those indicated by an asterisk, whose function has been as-
signed solely on the basis of genome annotation. Branch length indicates the number of inferred amino acid changes per position. †Although the monensin,
nanchangmycin, and nigericin TEs function as discrete enzymes (type II architecture), they play a type I role in the biosynthesis (e.g., release of the mature
[
52,53]
chain),
and correspondingly, clade with the type I PKS TE domains.
acid substrates from simple acyl chains to more complex
chain-extension intermediates, derivatized as both their N-ace-
tylcysteamine (NAC) thioesters and p-nitrophenylate (p-NP)
[17,34,35]
esters.
NAC has an identical structure to the substrate
end of the phosphopantetheine prosthetic group, and there-
fore NAC thioesters serve as mimics of the native carrier-pro-
tein-bound intermediates. In addition, as NAC is a poor leaving
group, these derivatives can be used to probe the relative
[17]
rates of acylation of the TE active site. However, we elected
to use the more reactive p-nitrophenylates in our assay for sev-
eral reasons. Most importantly, turnover of these substrates
can be monitored conveniently by UV/Vis spectroscopy (lmax
=
400 nm for p-nitrophenolate); this enabled us to assay all of
the TE domains in high-throughput fashion against four differ-
ent substrates. Secondly, benchmark data for these substrates
Figure 3. SDS-PAGE analysis of new TE domains expressed in this study. The
calculated molecular weights are as follows: AjuTE (32 kDa; lane 2), JerTE
[34]
[17]
with DEBSTEI and TylTEII were available in the literature;
this allowed independent verification of our results. Finally, de-
(33 kDa; lane 3), DisTEI (37 Da; lane 4) and SpiTEI (33 kDa; lane 6). Lanes 1
and 5 contain molecular weight markers.
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Promotion of Isochromanone Ring Formation
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
ꢀ1 ꢀ1
acylation of the acyl-enzyme intermediate has been shown to
83m s ; propionate 439m s ; butyrate 306m s ; valerate
ꢀ
1
ꢀ1 [17]
be the rate-determining step in turnover of the p-NP esters by
284m s ).
[
17,19,35]
both type I and II TEs.
As this is the stage at which the
In contrast, DEBSTEI did not exhibit Michaelis–Menten be-
havior towards p-NP acetate, propionate, and butyrate; instead
the kinetics showed evidence of substrate inhibition (Fig-
ure S7). Michaelis–Menten kinetics were obtained, however,
with 6 and DEBSTEI, consistent with the demonstrated prefer-
isochromanone ring would be formed in the normal pathway,
it was of particular interest to characterize TE substrate specif-
icity during this half of the reaction. One disadvantage of
using these esters, however, is that substrate-saturating condi-
tions cannot be attained due to their poor aqueous solubility,
and therefore it is not possible to determine apparent kcat and
KM values. Nonetheless, meaningful information has been ob-
tained by comparing the apparent specificity constants (kcat/
[34,36]
ence of the domain for longer acyl chains,
but the rate of
turnover remained modest relative to AjuTE, JerTE, and SpiTEI
with the same substrate (Table 1). No reaction was detected
between DisTEI and p-NP acetate, while propionate seemed to
inhibit the enzyme (Figure S8). Turnover of both butyrate and
valerate by DisTEI was substantially slower than that observed
for AjuTE, JerTE, and SpiTEI with these esters (Table 1).
One potential source of the inhibition seen with DEBSTEI
and DisTEI might be acylation of multiple nucleophilic residues
on the TE enzymes (e.g., cys-
K ) calculated from the initial rates of reaction at low concen-
M
[
17,19]
trations of various substrates.
All TEs were assayed in 96-well plate format with p-NP esters
–6 as substrates (Table 1). Based on preliminary experiments
3
to determine the limiting concentration of each substrate, the
teines) due to the high reactivity
Table 1. Kinetic analysis of the thioesterases. The TEs were assayed by using p-nitrophenyl esters 3–6.
of p-NP esters, as demonstrated
by mass spectrometry analysis
[17]
for TylTEII. Indeed, the sequen-
ces of both DEBSTEI and DisTEI
contain 2 and 3 cysteins, respec-
tively, that could be candidates
for modification. However, the
sequences of AjuTE, JerTE, and
SpiTEI also include a number of
Cys residues (2, 3, and 2, respec-
tively). Thus, to evaluate this
possibility directly, we monitored
for the acylation state of the TEs
ꢀ
1 sꢀ1
Substrate
Calculated apparent kcat/K
SpiTEI AjuTE
M
[m
]
DEBSTEI
DisTEI
JerTE
TylTEII
[
[
[
a]
a]
a]
[a]
3
4
5
6
n.d.
n.d.
n.d.
n.d.
n.d.
4.5ꢁ0.5
25ꢁ3
23ꢁ7
56ꢁ9
193ꢁ8
18ꢁ1
118ꢁ12
84ꢁ8
[a]
12ꢁ1
41ꢁ2
642ꢁ30
2.3ꢁ0.8
4ꢁ1
1024ꢁ75
1842ꢁ137
84ꢁ14
230ꢁ6
691ꢁ121
73ꢁ8
[a] Not determined due to substrate inhibition.
(
10 mg) by HPLC-MS, under con-
compounds were evaluated at six concentrations within the
solubility range (0.05–5 mm, depending on the substrate). For
each assay, the TE (1.3 mm) was incubated with one of the four
p-NP esters in assay buffer, and the rates of ester cleavage
were monitored at 400 nm. To obtain statistically significant
data, the rate of reaction at every substrate concentration was
monitored three times in duplicate, thus yielding six independ-
ent measurements for each ester. The rates of reaction were
then calculated from the initial linear portions of the curves,
and corrected for the background rates of chemical hydrolysis
of the p-NP substrates in the absence of the enzyme. For
AjuTE, JerTE, TylTEII, and the hydrolytic SpiTEI, the reaction
rates were proportional to substrate concentration for 3–6;
this enabled straightforward calculation of apparent k /K
ditions in which the p-NP propionate/protein ratios mimicked
those used in the kinetics assays (ca. 1800:1). This analysis re-
vealed that DEBSTEI and DisTEI were multiply acylated, and
that both domains always contained at least one label. In con-
trast, AjuTE and JerTE were modified with only a single propio-
nate group, or were unacylated; this is consistent with the lack
[17]
of inhibition. However, both SpiTE and TylTE whose activities
were not inhibited by propionate, also exhibited multiple acy-
lations. As there is no clear correlation between the level of
acylation and inhibition, the trends in activity could equally re-
flect a fundamental difference in behavior between the hydro-
lytic and the macrocyclization TE domains towards these
model substrates.
cat
M
values (Table 1, and Figures S3–S6). Among these TEs, JerTE
showed the highest reactivity towards all substrates, with a
clear preference for increasing chain length. The rate of re-
action of AjuTE and SpiTEI also rose with chain length, with
AjuTE showing consistently higher reactivity towards each sub-
strate, while TylTEII exhibited a moderate specificity for propio-
nate over the other p-NP esters. Reassuringly, although the
k /K values for TylTEII with each substrate were approximate-
Mutagenesis of ajuTE in Chondromyces crocatus Cm c5
We also probed the role of AjuTE in vivo by targeted mutagen-
esis of the domain. For this, we exchanged the active-site
Ser74 for an alanine (Figure 4A), as this mutation has been
[31]
shown to eliminate thioesterase activity effectively. Critically,
an equivalent replacement was engineered into DEBSTEI, and
[37]
shown not to disrupt the folding of the resulting protein.
cat
M
ly fourfold lower than the literature data, this experiment re-
produced the trend in relative reactivity seen earlier (acetate
The C. crocatus wild-type and two independently derived
ꢀ
ajuTE mutant strains were cultivated in triplicate and analyzed
by HPLC-MS. The yields of ajudazols in each case were com-
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1141

R. Mꢀller et al.
in the wild-type. Ajudazol B is obtained from the first-enzyme
free intermediate (Figure 1) by oxidation at C8 catalyzed by
[27]
the P450 AjuJ. Ajudazol A is generated by a two-step enzy-
matic process, in which the P450 AjuI acts first to install the
C15 exomethylene function (yielding deshydroxyajudazol A),
followed by AjuJ-catalyzed ring hydroxylation. Thus, one possi-
ble explanation for the drop in relative yield of ajudazol A is
that integration of the plasmid to generate the TE mutant
somehow reduced transcription of the adjacent ajuI gene.
It has been observed, albeit rarely, that disruption of the
[
14,39]
normal mechanism of product off-loading in PKS
and
[40,41]
mixed PKS/NRPS
systems, for example by frame-shift mu-
[40]
[41]
tagenesis or deletion of the TE domain, can lead to the
accumulation in culture extracts of chain-extension intermedi-
ates. Release of the substrates as their free acids does not
appear to be enzyme catalyzed, but instead to result from
[14]
spontaneous hydrolysis, presumably due to their extended
residency times on the ACP domains. However, despite careful
ꢀ
analysis by high-resolution HPLC-MS of extracts of ajuTE
[
40,41]
under conditions suitable for detection of carboxylic acids,
no such earlier intermediates of ajudazol biosynthesis were de-
tected. Thus, it appears that the only chain released spontane-
ously from the assembly line is that attached to the final ACP.
Discussion
The most notable structural feature of the natural product aju-
dazols of Chondromyces crocatus is the isochromanone ring, as,
[25]
along with the chromone of stigmatellin and the benzenoid
[26]
ring of lasalocid, it represents one of the few aromatic moi-
eties produced by a modular type I PKS/NRPS in bacteria. In
the case of the stigmatellin chromone, cyclization and aromati-
zation appear to be catalyzed by a novel type of cyclase
[
25]
Figure 4. Strategy for active-site mutagenesis of ajuTE in C. crocatus Cm c5.
A) Schematic of the ajudazol gene cluster showing the target gene ajuH,
and the mutagenesis plasmid pSUPHyginTEII. B) Genetic organization of the
mutant strain ajuTE containing integrated pSUPHyginTEII. PCR analysis (the
location of primers is shown) was used to confirm the presence of the
active-site mutant in the correct copy of the TE domain. C) Representative
domain located at the end of the multienzyme machinery. In
contrast, both the lasalocid and ajudazol assembly lines termi-
nate in thioesterase domains; this suggests potentially new
functions for these enzymes. Based on this premise, several
mechanisms can be proposed to account for formation of the
ꢀ
analysis by HPLC-MS (mixed extracted ion chromatogram of masses
[27]
ajudazol isochromanone (Scheme 2). In the first, the inter-
+
[
M+H] =591–594 and 647) of the production of ajudazols A (1) and B (2)
ꢀ
mediate is transferred to the TE active site Ser, followed by TE-
catalyzed attack of the C9 hydroxyl group onto the acyl termi-
nus, giving rise to a free ten-membered-ring lactone. Subse-
quent C2/C7 aldol addition and aromatization of ring I would
yield the isochromanone. Alternatively, aldol addition/aromati-
zation to form ring I occurs while the intermediate remains at-
tached to the TE domain, followed by TE-catalyzed lactoniza-
tion and chain release to yield ring II. The latter mechanism is
appealing, because the chain stays tethered to the TE until
ring formation is complete. Inspection of the structure of the
final intermediate suggests a third mechanism. Due to the
presence of the C4ꢀC5 cis double bond in the final intermedi-
ate, the chain is likely to adopt a configuration that would pro-
mote the C2/C7 aldol chemistry and attack by the C9 hydroxyl.
Analogous reactions have been shown to occur spontaneously
by C. crocatus wild-type (upper panel) and the ajuTE mutant (lower panel).
In this example, 1 and 2 were produced at 3% of wild-type levels, as judged
against chondramide A 7 as internal control. D) Mass spectra of 1 from the
+
+
analyses shown in (C). Ajudazol A [M+H] =591.3, [M+Na] =613.3.
pared to that of a third metabolite of the strain, chondrami-
[
38]
de A, on the assumption that the rate of chondramide bio-
synthesis would not be altered dramatically by modification of
the ajudazol gene cluster. Based on this internal control, the
ꢀ
combined yields of ajudazol A and B from the ajuTE inactiva-
tion mutant were reduced to 3–13% of wild-type levels (Fig-
ure 4C). The minor variations in production yields observed
with the mutants are typical for bacterial secondary-metabolite
producers, even when wild-type strains are grown in replicate
[
13]
[42]
under identical conditions.
on the reactive polyketone intermediates of the type II PKS,
Closer inspection of the data reveals that the amount of aju-
dazol B relative to ajudazol A was higher in the TE mutant than
and indeed the full functionality to form the isochromanone
ring is not installed until the last chain-extension cycle, thereby
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Promotion of Isochromanone Ring Formation
crete genes, the in silico analysis
also raised the possibility that
the ajuTE gene had once been a
stand-alone element, but had
become fused genetically to the
end of gene ajuH. In this case,
the covalent linkage of the TE to
the AjuH multienzyme (Figure 1)
would presumably have prevent-
ed the TE from carrying out its
normal proof-reading activity by
scanning along the multienzyme
assembly line, rendering it func-
tionally redundant. Indeed, the
His73 in the active-site motif of
AjuTE, which is highly conserved
in type II TE domains, has been
substituted with a serine; this
observation potentially indicates
that evolution is in the process
of inactivating the domain. To
probe these hypotheses directly,
we assayed AjuTE and a panel of
representative type I and II TEs
against four p-nitrophenylester
substrates.
Overall, the kinetics experi-
ment, the first to be carried out
with type II TEs from mixed sys-
tems, yielded two important ob-
servations relevant to the role of
the TE in isochromanone forma-
tion. The data showed that, de-
spite the absence of His73,
Scheme 2. Proposed mechanisms for the isochromanone ring formation and associated chain release in ajudazol
biosynthesis. A) The TE catalyzes attack of the C9 hydroxyl group on the acyl terminus to give rise to a free ten-
membered-ring lactone. Subsequent C2/C7 aldol addition and aromatization of ring I yield the isochromanone.
B) Aldol addition/aromatization occurs to form ring I, while the intermediate remains attached to the TE domain,
followed by TE-catalyzed lactonization and chain release to produce ring II. C) The ajudazol isochromanone moiety AjuTE is an active thioesterase
arises spontaneously by release from the ACP domain, with no involvement by the TE. D) Proposed function of
the jerangolid TE in catalyzing formation of a six-membered-ring lactone.
enzyme, and therefore that it
could potentially participate in
the off-loading reaction. Howev-
preventing premature chain release (Figure 1). Thus, it was also
a possibility that the ajudazol isochromanone moiety arises
spontaneously by release from the ACP domain, with no in-
volvement by the TE. Finally, we reasoned that the reaction
might be catalyzed on the ACP-bound intermediate by the in
trans action of a cyclase domain encoded outside the gene
cluster. Here we have used a combination of bioinformatic
analysis, enzymatic assay in vitro and in vivo gene inactivation
to try to discriminate among these models.
er, its efficient catalysis of chain release using water as a nucle-
ophile fits more closely with the behavior in our assay of the
type I and II hydrolytic TEs SpiTEI and TylTEII, than the type I
macrocyclization catalysts DEBSTEI and DisTEI (Table 1). Thus,
these kinetic data agreed with the idea that an ancestral TEII
gene might have been inadvertently fused to the ajudazol PKS,
and therefore that the resulting domain might not play a role
in the release of the polyketide chain. However, to investigate
the function of the TE in vivo directly, we inactivated the
domain by site-directed mutagenesis of the active-site serine.
This mutation was expected to stall the chain extension inter-
mediate on the ACP, as it could no longer be transferred to
Detailed sequence and phylogenetic analysis confirmed our
[
27]
preliminary observation
that, despite its type I (covalent)
context, AjuTE most closely resembles type II hydrolytic TE do-
mains from both PKS and NRPS systems (Figure 2). This con-
trasts with the TE domain of the lasalocid PKS, which clades
[40,41]
the TE domain.
ꢀ
Analysis of extracts of the AjuTE mutant revealed as much
as 13% of the wild-type levels of the ajudazols; this clearly
demonstrated that the isochromanone ring can form sponta-
neously in the absence of an active TE. Consistent with this
idea, the full-length chain is the only intermediate to be re-
leased from the PKS in detectable amounts when off-loading is
[17,19]
with typical type I TEs. Based on the literature precedent,
AjuTE would be expected to show a preference in the deacyla-
tion reaction for short acyl chains such as acetate and propio-
nate, consistent with its role in removing aberrant acyl units
from the carrier-protein domains. As TEIIs are encoded by dis-
ChemBioChem 2010, 11, 1137 – 1146
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1143

R. Mꢀller et al.
compromised. Nonetheless, the significant reduction in metab-
olite yields relative to the wild-type suggests that the TE accel-
erates this chemically favorable reaction when the intermedi-
ate is bound to its active site. Thus, even though the TE might
originally have functioned as a discrete, proof-reading domain,
it now appears to play an active role in synthesizing the iso-
chromanone ring. Indeed, the differences in substrate specifici-
PKS from the cyanobacterium Gloeobacter violaceus, in which a
type II TE again occupies a type I position. In future, it will be
interesting to identify the unknown product of this gene clus-
ter, so as to determine whether the reactivity pattern contin-
ues to hold.
ty between AjuTE, which shows a pronounced preference for Experimental Section
valerate, and “classic” type II TEs such as TylTEII, which typically
Strains and culture conditions: C. crocatus Cmc5 was grown at
favor propionate and butyrate over other chain lengths
[24]
3
08C and 170 rpm in liquid Pol 03 medium. Conjugation of the
[38]
[
17–20]
(Table 1),
could reflect a subtle adaptation of the domain
strain was performed as described previously, and mutants were
ꢀ
1
to the long-chain ajudazol structure. This finding allows us to
rule out two of the four mechanisms under consideration as
the principle mode of isochromanone ring formation (sponta-
neous release of the chain from the ACP, and participation of
an external cyclase), as neither of these means of release
should have been affected by inactivation of the TE domain.
Nonetheless, we cannot discriminate between the remaining
two mechanisms, as the precise way in which the TE promotes
isochromanone ring formation remains to be established. In
principle, the enzyme could use its catalytic machinery to acti-
vate the C9 hydroxyl group for attack on the acyl ester, instead
of water. Alternatively, the active-site environment might
simply help to channel the acyl terminus of the bound sub-
strate into a reactive conformation that favors spontaneous
cultivated in Pol03 containing hygromycin B (100 mgmL ). E. coli
DH10B and E. coli ET12567/pUB307 were grown in liquid LB
medium at 378C. When used with E. coli, antibiotics were present
at the following concentrations: hygromycin B (100 mgmL ), kana-
mycin sulfate (50 mgmL ), chloramphenicol (20 mgmL ), and am-
ꢀ1
ꢀ1
ꢀ1
ꢀ1
picillin (20 mgmL ).
General molecular biology methods: After gently crushing the
cell clumps with a glass homogenizer, C. crocatus Cm c5 chromoso-
[47]
mal DNA was prepared as described previously. Plasmid DNA
was isolated by using the GeneJET Plasmid Miniprep Kit (Fermen-
tas). DNA fragments were purified from agarose gels with the Nu-
cleoSpin Extract gel-extraction kit (Macherey–Nagel). Polymerase
chain reactions were carried out by using Taq DNA polymerase
(MBI Fermentas) or Phusion Polymerase (Invitrogen). DMSO was
added to the reaction mixtures to a final concentration of 5%.
Conditions for amplification with an Eppendorf Mastercycler Gradi-
ent Thermal Cycler were as follows: denaturation, 15–30 s at 95/
[
43]
ring closure and aromatization. Access of water to the active
site might be blocked by the presence of a longer than “stan-
dard” chain in the active site, and/or by the physical attach-
ment of the TE to the rest of the PKS multienzyme, thereby
9
88C; annealing, 20 s at 50–628C; extension, 15–60 s at 728C (30
cycles); final extension at 728C for 10 min. PCR products generated
for the construction of inactivation and expression plasmids were
verified by sequencing. All other DNA manipulations were per-
[
44]
suppressing competing hydrolytic release.
being relatively open, the active-site chambers of PKS and
Indeed, despite
[48]
formed according to standard protocols. DNA and amino acid
sequence analysis was performed with the Geneious 4.7.6 soft-
[
31,45]
NRPS type II TEs
are significantly shallower than the deep
[
30,46]
[28]
[49]
substrate tunnels of type I TEs;
this potentially helps to
ware package and ClustalW, and BLAST was used for compari-
son with GenBank data.
create the dehydrated conditions that promote cyclization.
The closest homologue to AjuTE in the public database is
Phylogenetic analysis of type I and II TE domains: 42 amino acid
sequences of type I and II TE from various biosynthetic systems
(PKS, NRPS, and hybrid PKS/NRPS) were retrieved from the NCBI
public database (http://www.ncbi.nlm.nih.gov/). The proteins were
[
29]
the TE domain from the jerangolid PKS, Like AjuTE, JerTE is
covalently linked to a PKS multienzyme (type I organization).
Consistent with their high mutual sequence identity, the do-
mains exhibited a similar pattern of substrate specificity to-
wards the simple acyl chains in our assays (Table 1), although
JerTE gave consistently higher rates of turnover. Intriguingly, in
its native context, the role of JerTE would be to catalyze forma-
tion of a six-membered ring by using an internal hydroxyl nu-
cleophile, a reaction directly analogous to the formation of
ring II within the isochromanone functionality (Scheme 2). As
with the isochromanone ring, generation of a six-membered-
ring lactone by release from an ACP in a model system derived
from the erythromycin PKS has been shown to occur sponta-
neously when the adjacent TE is inactivated by site-directed
[49]
aligned by ClustalW. Phylogenetic trees were created by using
neighborhood joining (NJ) as a distance method, as well as Bayesi-
an estimation; both programs were provided by Geneious Pro
[28]
4
.7.6.
For NJ, Jukes-Cantor was chosen as genetic distance
model, and bootstrapping was performed with 1000 replications.
The Bayesian inference method used the JTT-amino replacement
model and gamma distribution with four categories. Markov chain
Monte Carlo analysis (MCMC) was performed with 1.1 million gen-
erations and four independent chains, and the Markov chain was
sampled every 200 generations. Both methods gave similar tree
topologies.
Cloning of the TE expression constructs: Amplification of the
[
37]
[27]
[29]
[7]
[50]
mutagenesis. Thus, these data taken together seem to sug-
gest that AjuTE and JerTE are representative of a novel class of
type I TE that has evolved from type II TEs to accelerate the
biosynthesis of ring structures. This mechanism evokes the
function of cyclase domains in type II PKS systems, which
channel the spontaneous chemistry of reactive polyketone
genes for AjuTE, JerTE, SpiTEI, and DisTEI domains by PCR
was achieved by using the appropriate cosmid DNA. All forward
primers contained an artificial NdeI site (AjuTEexfwd (5’-GC CTG
CAT ATG CTC GGC GAG ACC CCC CCC GCG CGG AGC GCA-3’), Jer-
TEexfwd (5’-GG CTC CAT ATG CCC GCG GGG GAC GGA GAA GGA T-
3
’), SpirTEfwd (5’-TT GAG CAT ATG CAG GAC GAC ACG CAC GAC
GCG C)-3’), DisTEfwdI (5’-TG AGC CAT ATG AGC AAC GGC GCA GCC
CGG CA)-3’], whereas the reverse primers (AjuTEexrev (5’-CG AGG
GCG GCC GCT CAG AGC TGG CTG TGG ATC GA-3’), JerTEexrev (5’-
[
42]
chains towards a particular cyclic structure. This same strat-
egy appears to be in use a third time in an orphan modular
1144
www.chembiochem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1137 – 1146

Promotion of Isochromanone Ring Formation
GT CCA GCG GCC GCC TAG AGC TGG AGC GCG TGC TCG-3’), Spir-
TErev (5’-TTC GGC GGC CGC CTA CGC GGG GCG CGA GAG GAG-
enzyme, substrate, and DMSO was added to each well by using a
multichannel pipette (e1200, BioHit). Before measurement, the
plate was shaken for 90 s at 308C to allow thermal equilibration.
To obtain statistically relevant data, the entire assay was repeated
three times with each TE and with every substrate. The rates of hy-
drolysis were then calculated from the initial linear portion of the
curves and were corrected for the appropriate background rate of
chemical hydrolysis of the p-nitrophenyl derivatives. The apparent
k /K for each substrate was then obtained by calculating the
3
’), DisTErev (5’-AC CTG GCG GCC GCT CAT GAA AGC GCC TCG
CGG ACG TG-3’) were designed to introduce a NotI site down-
stream of the stop codon (restriction sites shown in bold). The PCR
products were cloned into pJET1.2 (Fermentas), digested with NdeI
and NotI, and subsequently ligated into the expression vector pET-
2
8b+ (Novagen), previously digested with NdeI and NotI. The
obtained expression constructs were designated as pET28b-AjuTE,
pET28b-JerTE, pET28b-SpiTE, and pET28b-DisTE.
cat
M
slope from a plot of n/[enzyme] versus [substrate].
i
Expression and purification of TE domains: Expression constructs
pET28b-AjuTE, pET28b-JerTE, pET28b-SpiTE, and pET28b-DisTE
were transformed into the strain E. coli Rosetta BL21 (DE3)pLysS/
RARE (Novagen). Expression was carried out in LB medium
Analysis of the acylation state of the TEs: Each TE protein (10 mg
ꢀ
1
ꢀ1
obtained from 10–20 mL of 1 mgmL and 0.5 mgmL solutions)
was incubated with p-nitrophenyl propionate (1–2 mL of a 0.5m so-
lution in acetonitrile) at 308C for 5 min. The reaction mixture was
then diluted with acetonitrile (20 mL) and water (20 mL) and acidi-
fied with formic acid (1 mL). The resulting solution was analyzed on
a CTultra ESI-MS ion trap instrument operating in positive-ioniza-
ꢀ1
(
200 mL) containing kanamycin sulfate (40 mgmL ) and chloram-
ꢀ1
phenicol (20 mgmL ) at 378C. Protein expression was induced at
A600 =0.8–1.0 by addition of isopropylthio-b-d-galactosidase (IPTG)
to a final concentration of 0.2 mm. After induction, the cells were
cultivated at 168C overnight, then harvested by centrifugation at
tion mode. Separation was achieved on a Jupiter C column (Phe-
4
ꢀ1
nomenex; 50ꢃ1 mm, 2.5 mm particle size, flow 0.3 mLmin ) with
a mobile phase consisting of water (A) and acetonitrile (B) each
containing 0.1% formic acid. The following gradient was applied:
1
5344g. Expression of TylTEII and DEBSTEI was performed as de-
[17,33]
scribed previously.
2
0–70% B over 3 min, 70–83% B over 8 min, 83–95% over 2 min.
Purification of all proteins was carried out at 48C by using an ꢂkta-
Prime Purification System (GE Healthcare). Cell pellets were resus-
pended in buffer A (20 mm Tris, pH 7.8, 200 mm NaCl, 10% glycer-
ol, 10 mm imidazole; 20 mL). The cells were broken by three
passes through a French Press (6.8948 MPa), and the insoluble ma-
terial was sedimented from the lysate by centrifugation at 15344g
and 48C. The lysate was filtered through a 1.2 mm syringe filter
Active-site inactivation of the ajudazol TE in C. crocatus Cm c5:
Site-directed mutagenesis of AjuTE was achieved by amplifying a
homologous inactivation fragment (1.5 kb) by overlap PCR using
oligonucleotides inTE2fwdI (5’-CGG TCC GAG CTT CCG CCT GGT
GC-3’), inTE2revI (5’-CG AGG AGG CCT CCC ATG GCG GAG CCG
AAC)-3’), inTE2fwdII (5’-GGC TCC GCC ATG GGA GGC CTC CTC GGC
TTC-3’), inTE2revII (5’-CCT GAG AGT GGC AGG GGT TGG GAG ACG-
3’). The nucleotides changed to achieve the active site Ser!Ala
mutation are shown in bold, and the artificial StuI restriction site
introduced for confirmation purposes is underlined (Table 1). The
final PCR product was cloned into pCR2.1TOPO (Invitrogen). The
fragment was then excised by using EcoRV and HindIII restriction
sites, and subcloned into pSUPHyg to generate the conjugation
plasmid pSUPHyginTEII, the sequence of which was verified by se-
quencing. Correct insertion of pSUPHyginTEII into the C. crocatus
genome was confirmed by PCR with the primers KPinTE4 (5’-CAG
CCT CCG TCA CCT C-3’) and pSUPEcoRV (5’-GCA TAT AGC GCT AGC
AGC-3’), followed by sequencing of the PCR product to demon-
strate the desired codon change in the appropriate copy of the TE
gene.
(
PALL), before being applied to a HisTrap HP column (1 mL; GE
Healthcare). All steps of the purification were carried out at a flow
ꢀ1
rate of 1 mLmin . The protein extract (20 mL) was loaded onto
the column after an equilibration step with buffer A (20 mL). After
loading, the column was washed with buffer A (20 mL), and then
the proteins were eluted by using a stepwise gradient with buf-
fer B (buffer A+500 mm imidazole) to give concentrations of 60,
[51]
1
00, 200, 300, and 500 mm imidazole. Elution of the proteins was
monitored by recording the absorbance at 280 nm. Appropriate
fractions were analyzed by SDS-PAGE. The fractions containing the
recombinant protein were pooled, concentrated with an Amicon
Ultra-4 concentrator (10 kDa cut-off; Millipore), and desalted into
assay buffer (200 mm potassium phosphate, pH 7.4, 10% glycerol)
by using a PD-10 column (GE Healthcare). Purified protein was
then flash frozen in liquid nitrogen and stored at ꢀ808C. Typically,
Analysis and quantification of secondary-metabolite production
by C. crocatus wild-type and ajuTE : C. crocatus Cm c5 and mu-
1
.7 mg of purified protein were obtained from 200 mL cell culture.
ꢀ
Determination of the kinetic parameters for TE-catalyzed hy-
drolysis: Hydrolysis of the p-nitrophenyl derivatives acetate, propi-
onate, butyrate, and valerate was monitored by following the rate
of p-nitrophenolate anion formation (lmax =400 nm) at 308C in 96-
well reaction plates by using a SpectraMax M5 (Molecular Devices)
plate reader. Reactions were carried out in buffer (200 mm potassi-
um phosphate, pH 7.4, 200 mL), containing TE protein (1.3 mm) and
variable concentrations of substrate in DMSO, and measured over
tants were grown in Pol03 medium containing 1% XAD adsorber
resin (Rohmer and Haas) for three days. The XAD beads and cell
clumps were harvested and extracted for 2ꢃ20 min by continuous
stirring with methanol. The extracts were evaporated and redis-
solved in methanol to give a 100-fold concentration of the original
culture volume. Standard analysis of crude extracts was performed
on an HPLC-DAD system from the Agilent 1100 series coupled to a
Brucker Daltonics HCTultra ESI-MS ion-trap instrument operated in
positive-ionization mode. Separation was achieved by using a Luna
RP-C18 column (Phenomenex; 125ꢃ2 mm, 2.5 mm particle size, flow
rate 0.4 mLmin ) with a mobile phase consisting of water and
acetonitrile each containing 0.1% formic acid, and with a gradient
from 5–95% acetonitrile over 20 min. Detection was carried out by
both diode array and ESI-MS. To enable quantification, mutant and
WT C. crocatus were cultured in triplicate, by inoculating each flask
with 2 g of wet cell mass. Cultivation and extraction were per-
formed as described above. Metabolite quantification was carried
out by using Bruker Daltonics QuantAnalysis 2.0. The relative
1
5
0 min. The overall DMSO content in each assay was adjusted to
%. Each substrate was measured at six different concentrations as
ꢀ1
follows: 0.5–5 mm for p-nitrophenyl acetate, 0.5–2.5 mm for p-ni-
trophenyl propionate, 0.25–1.25 mm for p-nitrophenyl butyrate,
and 0.05–0.25 for p-nitrophenyl valerate. A single experiment con-
sisted of measuring the reaction rate at each substrate concentra-
tion in the absence of the TE (six assays) to determine the back-
ground rate of chemical hydrolysis in the absence of enzyme, and
two assays for each of the substrate concentrations in the pres-
ence of the TE. To ensure uniformity, a mixture of ice-cold buffer,
ChemBioChem 2010, 11, 1137 – 1146
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1145

R. Mꢀller et al.
amounts of ajudazol A (1) and B (2) produced in each case were
judged against the yield of chondramide A as internal control. For
this, the amount of chondramide was calculated for each sample.
As the quantity of chondramide in each sample did not differ sig-
nificantly between the mutants and the wild-type, the yields were
averaged, and the mean value was assigned as 100%. The relative
amounts of 1 and 2 were then calculated with respect to this
value.
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·
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Published online on April 29, 2010
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