Polyketide proofreading by an acyltransferase-like enzyme

Article abstract of DOI:10.1016/j.chembiol.2012.01.005

Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for polyketide titer improvement and biosynthetic investigations.

Full text of DOI:10.1016/j.chembiol.2012.01.005

Chemistry & Biology
Article
Polyketide Proofreading
by an Acyltransferase-like Enzyme
Katja Jensen,1 Holger Niederkru¨ ger,1 Katrin Zimmermann,1 Anna L. Vagstad,2 Jana Moldenhauer,1 Nicole Brendel,3
1
1
1
2
4
3
¨
Sarah Frank, Petra Poplau, Christoph Kohlhaas, Craig A. Townsend, Marco Oldiges, Christian Hertweck,
1,
*
¨
and Jorn Piel
1
´
Kekule Institute of Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany
2Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA
3Leibniz Institute for Natural Product Research and Infection Biology, HKI, 07745 Jena, Germany
4Institute of Biotechnology 2, Forschungszentrum Ju¨ lich, 52425 Ju¨ lich, Germany
*Correspondence: joern.piel@uni-bonn.de
DOI 10.1016/j.chembiol.2012.01.005
SUMMARY
multiple covalently and noncovalently joined components (Piel,
2010). Trans-AT PKSs produce diverse bioactive polyketides,
Trans-acyltransferase polyketide synthases (trans- including the antibiotic bacillaene (1) from bacilli (Butcher et al.,
2007; Chen et al., 2006b; Moldenhauer et al., 2007) and the cyto-
toxins pederin (2) (Piel, 2002), psymberin/irciniastatin A (3)
(Cichewicz et al., 2004; Fisch et al., 2009; Pettit et al., 2004),
AT PKSs) are an important group of bacterial
enzymes producing bioactive polyketides. One
difference from textbook PKSs is the presence of
one or more free-standing AT-like enzymes. While
one homolog loads the PKS with malonyl units, the
function of the second copy (AT2) was unknown.
We studied the two ATs PedC and PedD involved in
pederin biosynthesis in an uncultivated symbiont.
and rhizoxin (4) (Iwasaki et al., 1986; Partida-Martinez and Hert-
weck, 2005) from various endosymbiotic bacteria (Figure 1).
Trans-acting components identified in these poorly understood
systems are ATs needed for loading the PKS with biosynthetic
building blocks (Cheng et al., 2003; Wong et al., 2011), enoylre-
ductases (ER) for reduction of a,b-unsaturated intermediates
PedD displayed malonyl- but not acetyltransferase
activity toward various acyl carrier proteins (ACPs).
(Bumpus et al., 2008), and enzymes to convert b-keto groups
to carbon branches (Calderone, 2008). The polyketide products
In contrast, the AT2 PedC efficiently hydrolyzed are almost (Musiol et al., 2011) exclusively constructed from
malonyl units, and a single, free-standing AT resembling those
from bacterial fatty acid synthases (here termed AT1) has been
shown to be sufficient for loading multiple trans-AT PKS modules
acyl units bound to N-acetylcysteamine or ACP. It
accepted substrates with various chain lengths and
functionalizations but did not cleave malonyl-ACP.
These data are consistent with the role of PedC
in PKS proofreading, suggesting a similar function
for other AT2 homologs and providing strategies
for polyketide titer improvement and biosynthetic
investigations.
with these units (Cheng et al., 2003; Lopanik et al., 2008). It
is therefore peculiar that more than half of all characterized
trans-AT PKS systems, including those of 1, 2, and 4, contain
another AT homolog (AT2). These enzymes are either free
standing or fused with the malonyl-loading AT and/or the
trans-ER. Except for the fact that their inactivation can result in
reduced polyketide titers (Lopanik et al., 2008), nothing is known
about the function of these ubiquitous enzymes.
INTRODUCTION
Much of the current knowledge about trans-AT PKS function-
ality stems from studies on the polyketide antibiotic bacillaene (1)
Bacterial complex polyketides are biosynthesized on giant multi- in Bacillus amyloliquefaciens and Bacillus subtilis (Bumpus et al.,
functional enzymatic assembly lines called modular polyketide 2008; Calderone et al., 2008, 2006; Chen et al., 2009; Dorrestein
synthases (PKSs) (Hertweck, 2009; Piel, 2010; Staunton and et al., 2006; Moldenhauer et al., 2007, 2010). Functional dissec-
Weissman, 2001). These enzymes contain numerous catalytic tion of the bacillaene (bae) PKS was greatly facilitated by the
domains, the order of which typically determines the structure discovery that mutants with an inactivated TE domain produce
of the polyketide chain. In addition, multiple acyl carrier protein polyketides of virtually all elongation stages (Moldenhauer
(ACP) domains serve as anchors that covalently bind intermedi- et al., 2007, 2010). This phenomenon—which has, with one
ates of increasing chain length via thioester linkages. After full exception (Yu et al., 1999), not been reported for textbook
elongation,
a C-terminal thioesterase (TE) domain usually (cis-AT) PKSs—suggests the existence of an as-yet uncharac-
cleaves the thioester bond by hydrolysis or lactonization to terized proofreading mechanism that releases stalled intermedi-
release the polyketide (Du and Lou, 2010). In textbook PKSs, ates from the enzyme. Such truncated products were also
all biosynthetic components needed to generate the polyketide observed for the rhizoxin (4) (Kusebauch et al., 2009) and, for
core structure are domains incorporated in the megaenzyme. some elongation stages, the mupirocin (Wu et al., 2008) path-
In contrast, the recently discovered group of trans-acyltransfer- ways, indicating that intermediate hydrolysis is a more general
ase (trans-AT) PKSs diverges from this architecture in using phenomenon for trans-AT PKSs. Here, we report genetic,
Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 329

Chemistry & Biology
Trans-AT PKS Proofreading
Figure 1. Selected Products of Trans-AT PKS
Pathways
O
HO
COOH
OH
Compounds are bacillaene (1), pederin (2), psymberin (3),
and rhizoxin (4).
HO HN
NH
O
1
O
OMe
OMe O
the influence of BaeB on thioester hydrolysis
could not be examined in this mutant. Therefore,
we created the double mutant JM157-54 that
lacked both the TE and BaeB to allow for direct
metabolic comparison with the TE single mutant
JM54-2. While JM54-2 produced the full series
of intermediates, only traces of two compounds
lacking three elongation units and one elonga-
tion unit, respectively, were detected in extracts
of the double mutant. This result may suggest
that BaeB is the off-loading factor. However,
we noticed that, for unknown reasons, all baeB
deletion mutants exhibited strongly diminished
growth. Thus, although relative polyketide yields
from both TE deletion strains had been normal-
ized to an internal standard (the PKS-indepen-
dent compound bacillibactin), we could not
exclude the possibility that the decreased
OMe
OH
OH
O
OH
O
MeO
O
H
H
N
MeO
N
3
OH
OH
O
O
OMe
O
OMe
2
O
O
O
HO
O
N
O
O
4
OMe
biochemical, and chemical studies of a member of the AT2 biosynthetic activity of the bae pathway was an indirect
group, identifying it as a thioester hydrolase for structurally effect of the altered physiology. This issue was addressed by
diverse N-acetylcysteamine- or ACP-bound acyl units. Our constructing a pHis8-based expression plasmid containing
data suggest that members of the AT2 group are not involved baeB for in vitro enzymatic studies. Expression resulted in
in acylation of PKS modules but act as proofreading factors soluble, N-terminally octahistidyl-tagged BaeB that was incu-
that reactivate PKS modules by releasing stalled acyl units.
bated with a range of N-acetylcysteamine (SNAC) conjugates
of short-chain carboxylic acids (5–9; Figure 2). SNAC thioesters
are known to be accepted as surrogates of ACP-bound interme-
diates by various PKS components (Kinoshita et al., 2001; Pohl
et al., 1998; Vergnolle et al., 2011). To detect thioester cleavage,
we monitored hydrolyzed free thiols in a continuous assay using
RESULTS
Study of BaeB, a Hydrolase Candidate from Bacillaene
Biosynthesis
Suspecting that the release factor is clustered with other PKS Ellman’s reagent (DTNB, 5,50-dithio-bis(2-nitrobenzoic acid)),
genes, we first focused on the previously uncharacterized which reacts with thiols to the yellow cleavage product 2-nitro-
gene baeB of the bacillaene (1) pathway. Its translated 5-thiobenzoate. However, no conversion was detected for any
product resembles thioester hydrolases, such as glyoxalase II, tested compound or condition.
and therefore represented a good candidate for intermediate
release. To investigate its function in vivo, we constructed the Differential Roles of the Pederin AT-like Enzymes
B. amyloliquefaciens mutant JM157 lacking baeB. Analysis of in Malonyl Transfer
the extracts by liquid chromatography-high resolution mass During these studies, it was found that TE deletion in the rhizoxin
spectrometry (LC-HRMS) showed that 1 was produced at signif- (3) producer Burkholderia rhizoxinica B1 resulted in a similar
icantly reduced (68%–87%) titers as compared to the wild-type release of intermediates as that known for bacillaene (Kuse-
strain FZB42 (Figure S1 available online), indicating that BaeB bauch et al., 2009). The fact that neither the rhizoxin (rhi) PKS
does not catalyze an essential biosynthetic step. Since no inter- cluster nor the entire genome (Lackner et al., 2011a, 2011b)
mediates are observed in strains harboring an intact TE domain, encoded a homolog of BaeB, combined with the negative results
Figure 2. SNAC Derivatives Tested in the Hydro-
lysis Assays
OH
O
O
O
H
N
H
H
N
N
S
S
S
For the preparation of compounds 6–9 see Experimental
Procedures. See also Figure S1.
O
O
O
O
5
6
7
O
O
O
H
N
H
N
H
N
S
S
S
O
O
8
9
10
330 Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Trans-AT PKS Proofreading
Figure 3. Phylogram of AT-like Enzymes from Various Trans-AT PKS Pathways
Tip labels consist of accession numbers, protein name, and domain architecture. For multidomain proteins, the relevant domain is shown in bold. Sequences from
the following pathways were used: Bae, bacillaene (B. amyloliquefaciens); Bat, batumin/kalimantacin (Pseudomonas fluorescens); Bry, bryostatin (‘‘Candidatus
Endobugula sertula’’); Dfn, difficidin (B. amyloliquefaciens); Dsz, disorazol (Sorangium cellulosum); Els, elansolid (Chitinophaga pinensis); Kir, kirromycin
(Streptomyces collinus); Lnm, leinamycin (Streptomyces atroolivaceus); Mmp, mupirocin (Pseudomonas fluorescens); Ozm, oxazolomycin (ADI12766 is from
Streptomyces bingchenggensis and ABS90474 from Streptomyces albus); Psy, psymberin (uncultivated symbiont of sponge Psammocinia aff. bulbosa); Rhi,
rhizoxin (Burkholderia rhizoxina); Sor, soraphen (Sorangium cellulosum). Outgroup is the E. coli AT from fatty acid biosynthesis (FabD).
of the BaeB hydrolysis assay, suggested that a different enzyme five AT-like genes within this strain complicates any in vivo
is responsible for thioester cleavage. The AT2 present in many knockout studies, we selected the AT homologs PedC and
trans-AT systems was a reasonable candidate because homo- PedD of the pederin (1) pathway from a Pseudomonas sp.
logs also occur in the bacillaene (1) and rhizoxin (4) pathways. symbiont of Paederus spp. beetles (Piel, 2002) for further
Moreover, thioester hydrolysis conforms to the mechanism of studies. These enzymes offered the distinct advantage of being
ATs, which involves transfer of a thioester acyl moiety to a serine among the very few known ATs that are not fused to additional
residue followed by nucleophilic displacement (Wong et al., domains, thus simplifying functional analysis.
2011). Since the bacillaene AT2 from B. amyloliquefaciens could In a phylogenetic analysis (Figure 3), PedD grouped with mem-
not be expressed as a soluble protein, and the presence of bers of the AT1 clade, including the previously characterized
Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 331

Chemistry & Biology
Trans-AT PKS Proofreading
used as test substrate, neither AT or ACP was labeled. These
results confirmed our in silico prediction that PedD is the AT
responsible for substrate loading and identified PedC as a candi-
date for intermediate release.
Malonyl-CoA
Acetyl-CoA
2
4
6
8
10 12 13 15 17 19 21
M
1
3
5
7
9 11 M 14 16 18 20 22
PedC
PedD
A
B
The AT2 PedC Hydrolyzes Acyl N-acetylcysteamine
Thioesters
A
A
A
PedI3 ACP
We next investigated hydrolysis of short-chain intermediate
mimics by PedC and PedD using the SNAC derivatives 5–9
and Ellman’s reagent. In agreement with its function as a malonyl
transferase, PedD did not have significant hydrolysis activity
toward any of the thioesters tested (Figure 5A). In contrast,
assays with the AT2 PedC showed conversion within minutes
for reactions with the acyl donors 5, 6, and 9. On the other
hand, hydrolysis was negligible for 7 and 8. We also tested
hydrolysis of the hexanoyl thioester 10 by removing aliquots at
different time points and quantifying the SNAC derivative after
high-pressure liquid chromatography (HPLC) separation. This
analysis revealed that 10 is hydrolyzed by PedC, albeit at a lower
rate than 5, 6, and 9 (Figure 5B). Expression levels of PedC were
very low, and even a 20 L culture provided just sufficient enzyme
amounts to determine kinetic parameters for substrate 5 (KM of
88.48 mM and a kcat/KM of 14.41 Mꢀ1sꢀ1) (Figure S4). These
should be regarded as preliminary, since SNAC derivatives
instead of PKS-bound acyl units were used and the true acyl
substrates could be fully elongated intermediates. All incuba-
tions with PedC also contained the chaperones GroEL/ES,
A
A
A
PedN ACP
PedC
PedD
PedI3 ACP
PedN ACP
Figure 4. Acyltransferase Assay Using the Pederin ATs
(A) SDS-PAGE analysis of incubations containing PedD (lanes 1–5, 13–17) or
PedC (lanes 6–8, 18–20), radiolabeled malonyl-CoA (lanes 1–12) or acetyl-CoA
(lanes 13–21), and the ACPs of PedI3 (tandem ACP) or PedN. ACPs were in the
holo form or the apo form, the latter indicated by an A. Protein markers are
indicated with an M.
(B) Autoradiogram of the same gel. See also Figure S2.
malonylating ATs LnmG of the leinamycin route (Cheng et al., from which the enzyme could not be separated (Figure S3). Prior
2003) and BryP (N-terminal domain) of the bryostatin pathway to kinetic characterization, background hydrolysis activity
(Lopanik et al., 2008). PedC fell into the AT2 group. To function- assays were therefore conducted using purified protein from
ally test the roles of ped ATs suggested by the phylogenetic data, parallel expressions containing the chaperones with and without
we produced both enzymes for in vitro analyses. Initial difficulties PedC coexpression. The full range of substrate concentrations
in obtaining soluble PedC by a range of expression strategies used for kinetic characterization were tested with both enzyme
were overcome by expressing the protein with an N-terminally purifications, where minimal background hydrolysis was
fused maltose-binding protein or a C-terminal Strep-tag in the detected in the protein preparation lacking PedC. These control
presence of the chaperones GroES and GroEL, which yielded experiments link the SNAC hydrolysis activity solely to PedC.
low amounts of soluble enzyme. PedC and PedD were then These results thus support a role for PedC as a hydrolase for
subjected to a transfer assay using [2-14C]-malonyl- or [1-14C]- acyl-SNAC thioesters carrying various functional groups that
acetyl-CoA as test substrate with an ACP as acceptor. For our typically occur during polyketide biosynthesis; i.e., b-hydroxyl
investigation of whether the two ATs might be specific for groups (2), a,b-double bonds (6), and a,b-reduced units (9,10).
different types of ACPs, the following proteins were expressed: It should be noted that the b-keto variant could not be tested,
the free-standing variant PedN (Piel et al., 2004a), the integrated since it was spontaneously hydrolyzed in the assay medium.
tandem domain PedI-ACP3 from module 3 of the pederin PKS Using the same assay, we also studied a PedC homolog from
(Piel et al., 2004b), and the integrated monodomain ACPs the rhizoxin (3) pathway. In this PKS system, the AT2 is fused to
PsyA-ACP3 and PsyD-ACP1 from elongation modules 3 and 4 a C-terminal AT1 on the bidomain protein RhiG (Partida-Martinez
of the psymberin PKS (Figure 4; Figure S2) (Fisch et al., 2009). and Hertweck, 2007). To uncouple both activities, three protein
PedN is expected to be involved in the generation of the exo- variants were expressed, in which none or either one of the AT
methylene carbon in pederin; PedI3 and PsyA3, to participate domains carried an inactivating Ser-to-Ala mutation at the acyl-
in the chain elongation process; and PsyD1, to belong to a non- binding site. However, neither protein displayed activity with
elongating module with an inactive ketosynthase domain. ACPs any of the SNAC derivatives 5–9. One reason for this unexpect-
were expressed in the apo form as well as in the holo form edly differential behavior of the ped and rhi AT2s might be that
carrying the 40-phosphopantetheinyl moiety that serves as inactive forms of RhiG were obtained during protein expression
anchor for the acyl units. After incubating the ATs, ACPs, and or purification. Since SNAC mimics were used instead of acyl-
radiolabeled acyl-CoA species in different combinations, we ACPs, we could not exclude the possibility that the observed
monitored the labeling of proteins by electrophoretic separation PedC activity was an artifact unrelated to PKS offloading.
and autoradiography. The analysis revealed that PedD self-loads
the malonyl radiolabel and efficiently transfers it to all types of The AT2 PedC Releases ACP-Bound Acyl Units
holo-ACPs but not to their apo forms. In contrast, ACPs re- This issue was addressed in a further series of experiments using
mained unlabeled in the PedC assay. With [1-14C]-acetyl-CoA ACP-bound 14C-radiolabeled acyl variants. Based on the ability
332 Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Trans-AT PKS Proofreading
Figure 5. Hydrolysis Assays Using Acyl-SNAC
and -ACP Test Substrates
A v [µM/min]
5
(A) Reaction rates observed for various SNAC derivatives.
Error bars specify standard deviations.
(B) Consumption of hexanoyl-SNAC by PedC. After
treatment of the thioester with PedC, aliquots were
removed and separated by HPLC. The diagram shows the
amount of hexanoyl-SNAC determined by manual inte-
gration at 232 nm at different reaction time points.
(C) Phosphor image of PksA acyl-ACP hydrolysis assays
with PedC. The signal is emitted from radiolabeled acyl-
ACP after electrophoretic separation of proteins. Acyl-
ACP (10 mM) was treated for 10 min with 0 (minus sign
indicates negative control), 5, 0.83, 0.14, 0.023, or
0.005 mM PedC. See also Figures S3 and S4.
4
3
2
1
0
PedC
PedD
GroEL/S
These acyl-ACP variants were individually incu-
bated with a serial dilution of PedC in a fixed
time point assay. Subsequently, aliquots from
incubations were separated by polyacrylamide
electrophoresis, and radiolabeled proteins
were visualized by phosphorimaging (Figure 5C).
The data clearly show that hexanoyl- and acetyl-
ACP are completely hydrolyzed within 10 min at
higher PedC concentrations while malonyl-ACP
remained intact.
compound
number
5
6
7
8
9
B
peak area [mAu*s]
8000
6000
4000
2000
DISCUSSION
Our studies on SNAC and ACP derivatives
demonstrate that PedC hydrolyzes structurally
diverse thioesters. Acyl units accepted as
substrates include completely reduced, a,b-un-
saturated, b-hydroxylated, and branched moie-
ties of chain lengths between two and at least
six carbon atoms. None of the tested
compounds were cleaved by PedD. Further-
more, PedD, but not PedC, exhibited malonyl
transfer activity. These data are consistent
with differential roles for the two AT homologs
in pederin biosynthesis, during which PedD
loads all ACPs with malonyl units, and PedC
acts as a proofreading enzyme that removes
stalled acyl units from blocked modules. In
agreement, PedC did not cleave malonyl-ACP,
0
0
50
100
time [min]
150
200
C
PedC
hexanoyl-ACP
acetyl-ACP
malonyl-ACP
of PedC to hydrolyze hexanoyl-SNAC (10), [1-14C]-hexanoyl- which is an essential precursor for polyketide biosynthesis.
ACP was selected as an initial test substrate. Hexanoyl is the As PedC does not display detectable AT activity, we pro-
native starter unit used in aflatoxin biosynthesis by Aspergillus pose to rename this enzyme acyl hydrolase (AH). PedC
spp. fungi (Minto and Townsend, 1997), thus allowing us to homologs occur in many trans-AT PKS systems (Piel, 2010), sug-
test an extended acyl unit with its cognate ACP. Moreover, since gesting that AH-mediated proofreading is widespread in these
this ACP is derived from a fungal iterative PKS, we hoped to pathways.
obtain insights into whether PedC would act on enzymes other
To date, AT-like editing enzymes are only known from few
than trans-AT PKSs. To generate the hexanoyl-loaded protein, type II PKSs, where they participate in the selection of non-
the N-terminally His6-tagged ACP monodomain from the itera- acetyl starter units (Tang et al., 2004). A different type of editing
tive megasynthase PksA of Aspergillus parasiticus was ex- enzyme is known for cis-AT PKSs (Heathcote et al., 2001) and
pressed in its apo form and loaded using [1-14C]-hexanoyl- nonribosomal peptide synthetases (Schwarzer et al., 2002; Yeh
CoA by the phosphopantetheinyl transferase Svp (Crawford et al., 2004). Here, free-standing (type II) TEs act as release
et al., 2006). In an analogous way, we also prepared [1-14C]- factors for the removal of stalled aberrant units. These units
acetyl- and [2-14C]-malonyl-ACP as further test substrates. are likely short-chain (amino-)acyl moieties that can arise from
Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 333

Chemistry & Biology
Trans-AT PKS Proofreading
premature decarboxylation of PKS extenders (Heathcote et al., hydrolyzes SNAC- and ACP-bound acyl units with various
2001) or from misloading of carrier proteins by either phospho- functional groups and chain lengths. In contrast, neither
pantetheinyl transferases (Schwarzer et al., 2002) or building the malonyl-transferring AT nor the bacillaene-specific
block-selecting domains (Yeh et al., 2004). In agreement, almost enzyme BaeB with an as yet unknown function displayed
all examined type II TEs preferred short-chain reduced acyl units measurable hydrolase activity in vitro. These results suggest
in in vitro assays, while more extended moieties and those the existence of a group of editing hydrolases, here termed
carrying hydroxyl or double bond functionality were poor or not AHs, that remove aberrant or stalled acyl units from blocked
substrates (Heathcote et al., 2001; Koglin et al., 2008; Schwarzer trans-AT PKS modules and might be responsible for inter-
et al., 2002; Yeh et al., 2004). This selectivity appears in contrast mediate release observed in several polyketide routes. The
to PedC, which cleaves acyl donors ranging from two (acyl-ACP) pederin AH or its homologs could be useful tools for
to six (9 and hexanoyl-ACP) carbon atoms and exhibits tolerance studying PKS functionality or, as inactivation of AHs has
for various functional groups occurring in polyketide biosyn- been shown to reduce polyketide titers (Lopanik et al.,
thesis. The data therefore suggest that PedC and other AHs 2008), for increasing compound yields by adding AH genes
might not only restore PKS modules blocked with decarboxy- to routes lacking them. In addition to attributing function
lated monomers but also play a role in the release of stalled to a core component of trans-AT PKSs, this study repre-
oligoketides. In agreement, all cases of intermediate release sents one of the few examples (Lopanik et al., 2008; Zimmer-
involving trans-AT PKSs, i.e., for bacillaene (Moldenhauer mann et al., 2009) that provide biochemical insights into
et al., 2007), rhizoxin (Kusebauch et al., 2009), and mupirocin polyketide pathways of uncultivated bacterial symbionts
(Wu et al., 2008), have been reported from assembly lines con- associated with invertebrates. As trans-AT PKSs are a major
taining AH-type enzymes. In trans-AT systems, this function group of enzymes responsible for natural product biosyn-
could be of particular significance since phylogenetic (Irschik thesis in these organisms (Piel, 2009), knowledge about their
et al., 2010; Nguyen et al., 2008; Teta et al., 2010) and preliminary enzymology is an important prerequisite for developing
unpublished functional data indicate that KSs are highly selec- heterologous expression systems for rare animal-derived
tive for the a,b-functionalization of incoming substrates. Thus, polyketides.
if b-processing is faulty, further elongation of incorrectly pro-
EXPERIMENTAL PROCEDURES
cessed intermediates would not occur, resulting in biosynthetic
blocks. This issue should be of less importance for cis-AT
PKSs, in which KS domains are rather promiscuous (Chen
et al., 2006a; Menzella et al., 2007). Nevertheless, hydrolysis of
long-chain intermediates was also reported for a small number
of cis-AT systems, an important example being rifamycin biosyn-
thesis (Yu et al., 1999). In this case, however, functional data
argued against an involvement of the type II TE, leaving the
underlying biochemistry unclear (Floss and Yu, 1999).
General
[1-14C]-hexanoyl-CoA, [1-14C]-acetyl-CoA, and [2-14C]-malonyl-CoA were
purchased from American Radiolabeled Chemicals, Perkin-Elmer or NEN
Radiochemicals, and Morovek, respectively. Unlabeled acyl-CoAs were
purchased from Sigma.
Bacterial Strains and Culture Conditions
Escherichia coli BL21(DE3) (Invitrogen) or E. coli Rosetta-gami 2 (DE3) pLysS
(Novagen) served as the host strain for protein expression experiments. E. coli
strain XL1 blue (Stratagen) served as host for routine subcloning. E. coli strains
were grown in Luria-Bertani (LB) medium (Sambrook and Russell, 2000).
Detailed information regarding antibiotics, additions, and inducer of the single
strains can be found in Table S1. Because it was not possible to obtain soluble
proteins without chaperones in some cases, pKJE7, pGro7, and pG-Tf2
(Takara) were used that produced DnaK-DnaJ-GrpE, GroEL-GroES, and
GroES-GroEL-Tig, respectively.
In conclusion, our data show that PedC, a member of a clade
of previously uncharacterized AT-like enzymes ubiquitously en-
coded in trans-AT PKS gene clusters, is a thioester hydrolase
that could play a key role in biosynthetic proofreading. Such
a function provides interesting perspectives for engineering
and studying PKS pathways. Addition of AHs to trans-AT PKS
pathways lacking these enzymes could result in enhanced poly-
ketide yields. With PKSs carrying engineered loss-of-function
mutations in their TE domains, AHs could be effective tools to
study entire PKS pathways by observing released intermediates
in a similar way as previously performed for bacillaene and
rhizoxin biosynthesis. Such potential applications are currently
under investigation in our laboratory.
Plasmids and General DNA Procedures
DNA isolation, plasmid preparation, restriction digests, gel electrophoresis,
and ligation reactions were conducted according to standard methods
(Sambrook and Russell, 2000). pBluescript II SK(+) was used for subcloning.
All primer sequences are listed in Table S2. Final constructs were verified by
sequencing. If not mentioned otherwise, PCR products were cloned into
pBluescript II SK(+) by using the method of Marchuk et al. (1991).
SIGNIFICANCE
Generation of Bacillus amyloliquefaciens Mutants and Polyketide
Analysis
Trans-acyltransferase polyketide synthases (trans-AT
PKSs) are an important but still poorly understood group
of biosynthetic enzymes involved in the production of bioac-
tive bacterial polyketides. About half of all known trans-AT
PKS gene clusters encode, in addition to the trans-acting
AT, another AT-like protein with a previously unknown func-
tion. We have identified for the first time an enzymatic
function for a member of this group. Our data show that
the pederin homolog from an uncultivated beetle symbiont
Generation of B. amyloliquefaciens strain JM54-2 (the TE single mutant) and
the conditions for polyketide analysis were published previously (Moldenhauer
et al., 2010). Sequences of PCR primers can be seen in Table S2.
To generate JM157-54 (lacking the TE and BaeB), we used a more complex
cloning strategy. A PCR product carrying the chloramphenicol resistance gene
was amplified from pDG268 (Antoniewski et al., 1990) using primers FP-cat-
BamHI and RP-cat-XbaI. This PCR product was cloned into pBluescript II
SK(+) to obtain pJM128. This plasmid was digested with XbaI/BamHI, and
the insert was ligated into the corresponding restriction sites of pJM135 to
obtain pJM140. To construct pJM135, a PCR product was amplified from
334 Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Trans-AT PKS Proofreading
B. amyloliquefaciens genomic DNA using primers RP-H-vor-Promotor-XbaI
and FP-H-vor-Promotor-NotI and ligated into pBluescript II SK(+) to yield
pJM133. This plasmid was digested with NotI/XbaI, and the resulting insert
was ligated into the corresponding restriction sites of pBluescript II SK(+)
to obtain pJM135. For the generation of pJM137, a PCR product was
amplified from B. amyloliquefaciens genomic DNA using primers RP-nach-
BaeB-2-KpnI and FP-nach-BaeB-2-ApaI. This PCR product was ligated
into pBluescript II SK(+) to obtain pJM137. pJM137 was then digested with
ApaI/KpnI, and the insert was ligated into pJM140 to obtain pJM155. A PCR
product was amplified from B. amyloliquefaciens genomic DNA using primers
RP-Promotor-ApaI and FP-Promotor-BamHI and ligated into pBluescript II
SK(+) to give pJM139. The insert of pJM139 was digested with BamHI/ApaI
and inserted into the corresponding site of pJM155 to obtain pJM157.
After linearization of pJM157 with KpnI, the DNA was integrated into the
genome of B. amyloliquefaciens JM161 (construction described later) by
double crossover to yield the baeB deletion mutant JM157. In the last step,
pJM54 (Moldenhauer et al., 2010) was linearized with SacI and transformed
into B. amyloliquefaciens JM157 to obtain the baeB-TE double mutant
JM157-54.
into pBluescript II SK(+) to obtain pKZ32. pKZ32 was then digested with BglII
and HindIII, and the 240 bp fragment was inserted into the corresponding site
of pHis8 svp to yield pKZ124. To construct the apo-PedN expression plasmid
pKZ161, pKZ124 was cut with NotI to remove the svp gene and then religated
to obtain pKZ161.
To construct the holo-PsyA expression plasmid pHN77, a 300 bp PCR
product was amplified from pPSKF1 (Fisch et al., 2009) using primers PsyA-
ACP3 for and PsyA-ACP3 rev. This PCR product was ligated into pBluescript
II SK(+) to obtain pHN75. pHN75 was then digested with BamHI and HindIII,
and the 300 bp fragment was inserted into the corresponding site of pHis8
svp to yield pHN77. To construct the apo-PsyA expression plasmid pHN60,
a 300 bp PCR product was amplified from pPSKF1 (Fisch et al., 2009) using
primers PsyA-ACP3 for and PsyA-ACP3 rev. This PCR product was ligated
into pBluescript II SK(+) to yield pHN59. pHN59 was then digested with
BamHI/EagI, and the 300 bp fragment was inserted into the corresponding
site of pHis8 to yield pHN60.
To construct the holo-PsyD expression plasmid pHN78, a 320 bp PCR
product was amplified from pPSKF1 using primers PsyD-ACP1 for and
PsyD-ACP1 rev. This PCR product was ligated into pBluescript II SK(+) to
obtain pHN76. pHN76 was then digested with BamHI and HindIII, and the
320 bp fragment was inserted into the corresponding site of pHis8 svp to
yield pHN78. To construct the apo-PsyD expression plasmid pHN66,
a 320 bp PCR product was amplified from pPSKF1 using primers PsyD-
ACP1 for and PsyD-ACP1 rev. This PCR product was ligated into pBluescript
II SK(+) to obtain pHN65. pHN65 was then digested with BamHI and EagI, and
the 320 bp fragment was inserted into the corresponding site of pHis8 to yield
pHN66.
The following strategy was used to generate JM161. A PCR product was
amplified from pUCemr (gift from R. Borriss, Humboldt University, Berlin,
Germany) using primers FP-erm-XbaI and RP-erm-ApaI and ligated into
pBluescript II SK(+) to yield pJM136. The plasmids pJM135 and pJM136
were both digested with XbaI and ApaI, and the insert of pJM136 was ligated
into pJM135 to obtain pJM143. pJM137 was digested with ApaI and KpnI, and
this insert was ligated into the corresponding restriction sites of pJM143 to
obtain pJM145. The erythromycin antibiotic cassette was replaced with the
pIC333 (Steinmetz and Richter, 1994) spectinomycin cassette through a
blunt-end/XbaI ligation into pJM145 to obtain pJM161. The resulting construct
was linearized and transformed into the B. amyloliquefaciens wild-type strain
FZB42 to obtain strain JM161.
Construction of the expression plasmid for the PksA ACP was reported
earlier (Crawford et al., 2006).
The gene coding for RhiG was amplified by PCR from cosmid 20G04
(Partida-Martinez and Hertweck, 2007) with the primers Expr_tAT_F and
Expr_tAT_R. Ligation into the EcoRV site of the pGEM-T Easy cloning vector
(Promega) yielded pNB118. The NdeI/HindIII fragment of pNB118 was ligated
into the NdeI/HindIII sites of pET28a(+) expression vector, which carries an
N-terminal His6-Tag, resulting in pNB121.
Construction of Expression Plasmids
All primer sequences to generate inserts of expression plasmids are provided
in Table S2.
To introduce point mutations into active site of RhiG, we used the
QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) on pNB121
(AT1ꢁAT2–S106A and AT1AT2ꢁ–S468A). The plasmid pNB146 contains
a rhiG variant coding for AT1ꢁAT2, and the plasmid pNB147 contains a rhiG
variant coding for AT1AT2ꢁ.
To construct the BaeB expression plasmid pKJ7, a 0.7 kb PCR product was
amplified from B. amyloliquefaciens genomic DNA using primers BaeB_low-
GC_pHis8-3_for and BaeB_low-GC_pHis8-3_rev. This PCR product was
ligated into pBluescript II SK(+) to obtain pKJ1. pKJ1 was then digested with
BamHI and HindIII, and the 0.7 kb fragment was inserted into the correspond-
ing sites of pHis8 (Jez et al., 2000).
To generate the pedC C-terminal strep-tagged fusion construct, a 1 kb PCR
product was amplified from pPD23E7 (Piel, 2002) using primers pedC_FP and
pedC_RP. This PCR product was ligated into pBluescript II SK(+) to obtain
pHN37. pHN37 was then digested with BsaI, and the 1 kb fragment was in-
serted into the corresponding site of pASK-IBA3 (IBA Biotagnology) to yield
pHN38.
Expression and Purification of Proteins
To prepare sufficient amounts of PedC for kinetic studies, large-scale fermen-
tation was needed. A colony of E. coli cells freshly transformed with pHN38
and pGro7 was cultivated up to an optical density (OD600) of about 4 at 37ꢁC
in 2 ml LB medium. The grown biomass was used to inoculate the second pre-
culture in 1 l baffled shake flasks with 150 ml culture volume at 120 rpm minꢀ1
and 37ꢁC. The second preculture medium contained 5 g/lꢀ1 yeast extract,
10 g/lꢀ1 tryptone, 2.5 g/lꢀ1 NaCl, 0.25 g/lꢀ1 MgSO4 $ 7 H2O, 1.26 g/lꢀ1
NaH2PO4, and 10 g/lꢀ1 glucose, adjusted to pH = 7.2 with 4 N NaOH. Main
cultivation was performed in a 30 l scale bioreactor (Chemap) with 20 l initial
culture volume at 37ꢁC. Dissolved oxygen concentration was controlled
at R40% of air saturation, and the pH was kept at 7.0 with 25% ammonia solu-
tion. Medium was similar as for the second preculture, except that 20 g/lꢀ1
glucose and 5 g/lꢀ1 arabinose were present. A total of 900 ml of the second
preculture was used to inoculate the main culture, resulting in an initial
OD600 = 0.25. At OD600 = 4.5 (after about 3 hr), 4 mg of anhydrotetracycline
(IBA Biotagnology) was added (resulting in 200 ng/ml), and the temperature
was decreased to 25ꢁC in less than 10 min. After 7 hr cultivation time, a glucose
feed (600 g/lꢀ1 glucose monohydrate) was started to keep the glucose
concentration in a range of 13–17 g/l throughout the cultivation process.
Glucose measurement was performed using an AkkuCheck glucose analyzer
(Roche Diagnostics). After 24 hr of cultivation time, the bioreactor was cooled
to 10ꢁC, and the cell suspension (OD600 = 35) was harvested. Biomass sepa-
ration was achieved by centrifugation at 8,000 rpm (rotor type JLA8.1000,
Avanti J-20 XP, Beckman), and the biomass was stored at ꢀ80ꢁC prior to
further protein purification. The wet cell weight was 500 g. For kinetic measure-
ments of PedC, elution fractions were dialyzed three times for at least 3 hr
The pedC C-terminal hexahistidine-tagged fusion construct pET-pedC was
generated by PCR amplification of the gene using pHN38 as template DNA
and pedC-5 and pedC-3 as primer. The PCR product was ligated into pET-
24a(+) (Novagen) at the NdeI and XhoI restriction sites after digestion of the
insert with the same enzymes.
To construct the PedD expression plasmid pKZ178, a 1.1 kb PCR product
was amplified from pPD23E7 (Piel et al., 2004b) using primers pedD_FP and
pedD_RP. This PCR product was ligated into pBluescript II SK(+) to obtain
pKZ177. pKZ177 was then digested with EcoRI and HindIII, and the 1.1 kb
fragment was inserted into the corresponding sites of pHis8 to yield pKZ178.
To construct the holo-PedI3 expression plasmid pKZ123, a 550 bp PCR
product was amplified from pS9D2 (Piel et al., 2004b) using primers ped
I3_pRSETf and pedI3_pRSETr. This PCR product was ligated into pBluescript
II SK(+) to obtain pKZ57. pKZ57 was then digested with BglII und HindIII, and
the 550 bp fragment was inserted into the corresponding site of pHis8 svp to
yield pKZ123. To construct the apo-PedI3 expression plasmid pKZ176,
pKZ123 was cut with NotI to remove the phosphopantetheinyl transferase
gene svp and then religated to obtain pKZ176.
To construct the holo-PedN expression plasmid pKZ124, a 240 bp PCR
product was amplified from Paederus fuscipes metagenomic DNA using
primers pedN_pRSETf and pedN_pRSETr. This PCR product was ligated
Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 335

Chemistry & Biology
Trans-AT PKS Proofreading
against 200 mM potassium phosphate buffer (KPi), pH 7.4 containing 10%
(v/v) glycerol. If necessary, fractions were concentrated with Vivaspin columns
(VWR). Protein concentration was determined either by Bradford assay or,
if expressed with chaperones, with SDS-PAGE-absorption measurements.
Specifically, SDS-PAGE was performed with all protein samples along with
BSA samples of known concentration. The band intensities were integrated
using the program GeneTools (Syngene). A calibration curve was established,
and the concentration of the protein samples was determined. Proteins were
flash frozen in liquid nitrogen after adding 10% (v/v) glycerol and stored
at ꢀ80ꢁC.
tiate between self-loading of ACPs and AT-mediated loading, we also per-
formed measurements without AT.
Acyl-SNAC Hydrolysis Assays
After desalting in 200 mM KPi, pH 7.4, and 10% (v/v) glycerol, proteins were
incubated with different test substrates at a range of concentrations. In these
assays, PedC was used only as purified, Strep-tagged protein (Figure S3). A
typical assay, adapted from Heathcote et al. (2001), consisted of 250 nM
AT, 200 mM KPi, pH 7.4, 0.2 mM DTNB (Alfa Aesar), and 3% (v/v) dimethylsuf-
oxide (DMSO) at 30ꢁC in a total volume of 1 mL. All test substrates were dis-
solved as 2 M stocks in DMSO except compound 5, which was dissolved in
200 mM KPi, pH 7.4. A 20 mM DTNB stock solution was resuspended in
200 mM KPi, pH 7.4. Samples containing 7 and 9 were incubated for 2 min
at room temperature (RT) prior to measurement, allowing better solubilization.
Enzymatic activities were monitored by measuring the absorbance increase at
For the overexpression of all other proteins, except for pET-PedC, 1 l
cultures were grown to an OD600 of 0.7 (37ꢁC, 200 rpm), induced with either
isopropyl b-D-1-thiogalactopyranoside (IPTG) or anhydrotetracycline, and
grown for approximately 20 hr at 16ꢁC. Cells of expression strains were
harvested by centrifugation (11,000 3 g, 7 min) at 4ꢁC. For pET-PedC, 1 l
expression cultures were supplemented with 50 ng/mlꢀ1 tetracycline prior to
inoculation with a 10 ml saturated starter culture. Cells were grown at 37ꢁC
250 rpm, and growth was followed by monitoring OD600. The cultures, at
OD600 ꢂ0.4, were cooled on ice for 15 min, induced with 1 mM IPTG, and
expressed at 18ꢁC for 16 hr.
412 nm resulting from hydrolysis of Ellman’s regent (ε = 14,150 Mꢀ1 cmꢀ1
)
(Frank et al., 2007) for 18 min using a BioMate 3 photometer (Thermo). All
measurements were carried out in at least duplicate and corrected for the
background rate of hydrolysis without enzyme. For the determination of kinetic
parameters, initial rates were plotted versus the substrate concentration and
fitted to the Michaelis-Menten equation with GraphPad Prism 4.0 (GraphPad
Software). For those enzymes, a Michaelis-Menten fit was not possible; only
the initial velocities were plotted.
Cells expressing proteins with His6- or His8-Tag were resuspended in lysis
buffer (25 mM Tris-HCl, pH 7.6, 0.5 M NaCl, 10 mM imidazole, and 0.1%
(v/v) Triton X-100). The pH was adjusted to 7.6 in the case of PedD, RhiG,
and their derivatives. A pH of 8 was chosen for all other proteins. The cell
suspension was kept on ice and sonicated in 10 s intervals. The cell debris
was removed by centrifugation at 23,500 3 g for 30 min at 4ꢁC. The superna-
tant was incubated with 1.0 ml Ni-NTA agarose (QIAGEN) for at least 1 hr at
4ꢁC and washed with 10 ml lysis buffer. His-tagged proteins were eluted
with lysis buffer containing increasing concentrations of imidazole up to
250 mM. For the expression using pET-pedC, a modified protocol was used.
Cells were harvested by centrifugation (4,100 3 g, 20 min) at 4ꢁC, resus-
pended (1 g per 4 ml) in Ni-buffer (50 mM KPi pH 7.6, 300 mM NaCl, 10% glyc-
erol, 20 mM imidazole), and lysed by treatment with 1 mg/ml lysozyme (Sigma)
for 30 min, followed by sonication in 10 s intervals. The lysate was clarified by
centrifugation (27,000 3 g, 30 min), decanted, and bound to 1 ml Ni-NTA resin
(QIAGEN) for 1 hr rotating at 4ꢁC. The resin was washed three times with 10 ml
Ni-buffer and eluted three times with 0.5 ml Ni-buffer containing 250 mM imid-
azole. Fractions were analyzed by SDS-PAGE. The flowthrough contained
a large amount of unbound PedC, which was subsequently collected by
a second round of Ni2+ affinity chromatography. Fractions containing PedC
were pooled, concentrated to ꢂ7 mg/mlꢀ1 using a 10,000 MWCO Amicon
Ultra Centrifugal Filter Unit (Millipore), and dialyzed against storage buffer con-
taining 100 mM KPi pH 7.0, 10% glycerol, and 2 mM dithiothreitol (DTT). The
concentration of PedC was determined by Bradford assay in duplicate and
adjusted for GroEL content.
Hydrolysis of hexanoyl-SNAC (mimicking the hexanoyl starter unit of PksA
from Aspergillus parasiticus involved in norsolorinic acid biosynthesis)
was determined using an HPLC-based assay. Hexanoyl-SNAC (1 mM) was
treated with 5 mM PedC in 100 mM KPi, pH 7.0, 10% glycerol, and 1 mM
DTT. At each time point, 150 ml reaction aliquots were passed through
a 10,000 MWCO Microcon centrifugal filter device (Millipore) at 14,000 3 g
for 10 min to remove the protein. Fifty microliter injections of microcon
˚
flowthrough were separated over a Prodigy 5m ODS3 100 A, 250 3 4.6 mm
column (Phenomenex) using an Agilent Technologies 1200 series HPLC and
a 40:60:0.1 acetonitrile:water:formic acid isocratic method at 1 ml/minꢀ1
.
The peak area of hexanoyl-SNAC (RT for 12.0 min) was determined by manual
integration at 232 nm wavelength. Controls with no enzyme or BSA showed
no loss in hexanoyl-SNAC signal, indicating that hydrolysis was catalyzed
by PedC.
Acyl-ACP Hydrolysis Assays
To determine whether PedC can hydrolyze acyl-ACP intermediates, we used
a radiochemical hydrolysis assay. The PksA ACP monodomain was activated
with various 14C-labeled CoAs and treated with a serial dilution of PedC in
a fixed time point assay.
The N-terminally hexahistidine-tagged PksA apo-ACP monodomain
(excised from the six domain iterative megasynthase) was purified as previ-
ously described and activated by an amended protocol described later (Craw-
ford et al., 2006). 14C-labeled acyl-ACP was generated by treating 100 mM
apo-ACP with 5 mM Svp (phosphopantetheinyl transferase from Streptomyces
verticillus), and 400 mM total acyl-CoA (mixture of unlabeled and 14C-labeled to
achieve 5.5 mCi/mlꢀ1) in 100 mM KPi, 10 mM MgCl2, 1 mM TCEP, and 10%
glycerol for 45 min at RT. Acyl-ACP can be frozen for later use or diluted to
be used immediately.
Cells containing expression constructs with Strep tags were resuspended in
Buffer W (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA). After sonica-
tion and centrifugation as described earlier, the supernatant was applied to
a column with 1 ml Strep-Tactin-Sepharose (IBA Biotagnology). The column
was washed twice with 5 ml Buffer W and eluted with six fractions of 0.5 ml
Buffer E (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM des-
thiobiotin). Columns were used up to five times and regenerated according
to manufacturer’s specifications.
PedC was thawed and serially diluted (1:5) to five different concentrations
ranging from 20 to 0.02 mM 43 stock solutions in 100 mM KPi, pH 7.0, with
10% glycerol. Twelve microliters of each PedC stock was pre-aliquoted, and
the assay was initiated by addition of 36 ml of 13.3 mM 14C-acyl-ACP. After
10 min at room temperature, 20 ml aliquots of each reaction were quenched
into 10 ml of 43 SDS loading dye. Final reaction conditions were 10 mM acyl-
ACP treated with 0 (negative control), 5, 0.83, 0.14, 0.023, or 0.005 mM
PedC. Samples were separated by 16% SDS-PAGE, dried on filter paper
with a slab gel dryer (Hoeffer Scientific), exposed overnight to a storage phos-
phor screen (Amersham), and imaged with a Typhoon 9410 Imager (Johns
Hopkins Integrated Imaging Center).
Acyltransferase Assays
To study acyl transfer from ATs to ACPs, we performed assays with [2-14C]-
malonyl-CoA and [1-14C]-acetyl-CoA (Perkin-Elmer). Assay mixtures con-
sisted of 4 ml DTT (10 mM), 5 ml AT, 9 ml ACP, and either 2 ml [malonyl-2-14C-
CoA] (0.01 mCi/mlꢀ1) or 1 ml acetyl-CoA [acetyl-1-14C-CoA] (0.02 mCi/mlꢀ1).
Lysis buffer, instead of buffer containing the enzyme, was used in the negative
controls. Everything was mixed on ice, and the reaction was started with the
addition of the substrate. Samples were incubated on ice for various times, fol-
lowed by the addition of equal volumes of 23 SDS-sample buffer (0.09 M Tris-
HCl, pH 6.8, 20% (v/v) glycerol, 2% (v/v) SDS, 0.02% (v/v) bromophenol blue,
0.1 M DTT) and incubation at 99ꢁC for 3 min. After separation by SDS-PAGE,
the gels were dried and exposed to X-ray film. Band quantification was per-
formed with an Image Reader and the software Bas Reader (Raytest). The
autoradiogram was edited with the software TINA 2.09d (Raytest). To differen-
Phylogenetic Analysis
Sequences of AT-like proteins were aligned using MUSCLE. Phylogenetic
analysis was performed by maximum likelihood inference using PhyML with
a Jones-Taylor-Thornten amino acid replacement model.
336 Chemistry & Biology 19, 329–339, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Trans-AT PKS Proofreading
Synthesis of Acyl-SNAC Derivatives 5–10
S.F., P.P., and C.K. synthesized SNAC derivatives; and J.P. performed the
phylogenetic analysis. All authors designed the experiments, analyzed the
data, and wrote the manuscript.
Synthesis of the known compounds 5 and 10 was performed as published
previously (Crawford et al., 2006; Gru¨ schow et al., 2007) The general
procedure for compounds 6–9 was as follows. A solution of the corresponding
acid (5 mmol) in dichloromethane (15 ml) was cooled down to 0ꢁC for 15 min.
Received: October 18, 2011
Revised: December 9, 2011
Accepted: January 2, 2012
Published: March 22, 2012
4-Dimethylaminopyridine (DMAP,
1 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide (EDC, 6 mmol), and N-acetylcysteamine (6 mmol) were
added, and the mixture was stirred overnight at 25ꢁC. The reaction mixture
was quenched with saturated aqueous ammonium chloride and extracted
with dichloromethane. The combined organic phases were dried with
MgSO4, filtered, concentrated, and subjected to silica gel column chromatog-
raphy as eluant to provide the respective thioester.
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Yield was as follows: 54%, column chromatography with ethylacetate. 1H-
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3
3
4
6.9 Hz), 6.16 (dq, 1H, JH-H = 15.4 Hz, JH-H = 1.6 Hz), 5.95 (brs, 1H), 3.46
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4
3JH-H = 6.9 Hz, JH-H = 1.6 Hz); 13C-NMR: (100 MHz, CDCl3, RT) d [ppm] =
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190.5, 170.6, 142.1, 130.1, 40.1, 28.4, 23.4, 18.2; MS: (ESI, 10 eV, m/z (%))
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3
=
3
15.5 Hz, JH-H = 7.0 Hz), 6.13 (td, 1H, JH-H = 15.5 Hz, JH-H = 1.5 Hz), 5.88
3
4
3
3
(bs; 1H), 3.49-3.44 (pq, 2H, JH-H = 6.5 Hz), 3.09 (t, 2H, JH-H = 6.5 Hz), 2.19
4
3
3
(ddt, 2H, JH-H = 1.5 Hz, JH-H = 7.0 Hz, JH-H = 7.3 Hz), 1.96 (s; 3H; H-1),
1.50 (tq, 3H, JH-H = 7.4 Hz), 0.94 (t, 6H, JH-H = 7.4 Hz); 13C-NMR:
(100 MHz, CDCl3, RT) d [ppm] = 190.4, 170.2, 146.5, 128.4, 39.8, 34.2, 28.2,
23.2, 21.2, 13.7; MS: (ESI, 10 eV, m/z) 120.0 (8) [NAC + H]+, 216.1 [M + H]+,
238.1 [M + Na]+.
3
3
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3
3
(q, 2H, JH-H = 6.0 Hz), 3.04 (t, 2H, JH-H = 6.0 Hz), 2.13 (s, 3H), 1.93 (s, 3H),
1.86 (s, 3H); 13C-NMR: (100 MHz, CDCl3, RT) d [ppm] = 189.8, 170.5, 155.1,
123.2, 40.2, 28.6, 27.5, 23.4, 21.5; MS: (ESI, 10 eV, m/z (%)) 210.1 (100)
[M + Na]+; HR-MS (ESI, 10 eV, m/z): C9H15NO2SH 202.0896; measured:
202.0893 [M + H]+.
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(m, 2H), 1.96 (s, 3H), 1.62-1.53 (m, 3H), 0.90 (d, 6H); 13C-NMR: (100 MHz,
CDCl3, RT) d [ppm] = 200.3, 170.2, 42.2, 39.7, 34.3, 28.4, 27.6, 23.2, 22.2;
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Crawford, J.M., Dancy, B.C.R., Hill, E.A., Udwary, D.W., and Townsend, C.A.
(2006). Identification of a starter unit acyl-carrier protein transacylase domain
in an iterative type I polyketide synthase. Proc. Natl. Acad. Sci. USA 103,
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Supplemental Information includes four figures and two tables and can be
found with this article online at doi:10.1016/j.chembiol.2012.01.005.
Dorrestein, P.C., Bumpus, S.B., Calderone, C.T., Garneau-Tsodikova, S.,
Aron, Z.D., Straight, P.D., Kolter, R., Walsh, C.T., and Kelleher, N.L. (2006).
Facile detection of acyl and peptidyl intermediates on thiotemplate carrier
domains via phosphopantetheinyl elimination reactions during tandem mass
spectrometry. Biochemistry 45, 12756–12766.
ACKNOWLEDGMENTS
We thank H. Chang, M. Engeser, C. Sondag, and H.-J. Brandt for technical
assistance; R. Borriss, B.S. Moore, and M. Marahiel for plasmids; and M.F.
Freeman for experimental suggestions. This work was financially supported
by the DFG (German Research Foundation) (PI 430/1-3, PI 430/6-1, PI 430/
9-1, SFB 624, and GRK 804 to J.P.).
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pathway; K.Z. and H.N. conducted the acyl transfer assays; K.J. conducted
acyl-SNAC hydrolysis assays; A.L.V. conducted acyl-ACP hydrolysis assays;
Fisch, K.M., Gurgui, C., Heycke, N., van der Sar, S.A., Anderson, S.A., Webb,
V.L., Taudien, S., Platzer, M., Rubio, B.K., Robinson, S.J., et al. (2009).
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