Substrate specificity in ketosynthase domains from trans-AT polyketide synthases

Article abstract of DOI:10.1002/anie.201207690

Branching out: The substrate specificity profiles for a range of ketosynthase (KS) domains from trans-AT PKSs are reported. Evidence is provided that a sterically demanding amino acid residue adjacent to the active-site Cys residue confers specificity towards non-β-methyl-branched substrates (see scheme). Copyright

Full text of DOI:10.1002/anie.201207690

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DOI: 10.1002/anie.201207690
Polyketide Biosynthesis
Substrate Specificity in Ketosynthase Domains from trans-AT
Polyketide Synthases**
Matthew Jenner, Sarah Frank, Annette Kampa, Christoph Kohlhaas, Petra Pçplau,
Geoff S. Briggs, Jçrn Piel,* and Neil J. Oldham*
Type I modular polyketide synthases (PKSs) catalyze the
production of a remarkable array of biologically active
natural products, with many employed as antibiotics, immu-
nosuppressants, anti-parasitics, and anti-tumor agents.[1] Each
PKS module possesses the enzymatic machinery for a single
cycle of chain elongation and modification. Within a module,
the acyltransferase domain (AT) loads an acyl-CoA deriva-
tive onto the phosphopantetheinyl (PPant) chain of the acyl
carrier protein (ACP). Claisen thioester condensation, cata-
lyzed by the ketosynthase (KS) domain, then results in chain
elongation. The extent of b-keto processing is dictated by the
presence of ketoreductase (KR), dehydratase (DH), and
enoylreductase (ER) domains allowing production of hy-
droxyl, olefinic, and fully saturated intermediates.[2]
Post-genomic techniques, and the propensity for type I
PKS genes to arrange into distinct modules, have allowed the
prediction, with fairly high confidence, of biosynthetic
schemes from genetic sequences alone.[3] However, recently
a novel group of PKSs lacking integral AT domains has been
reported that deviates from standard colinearity rules. In
these systems, the AT activity is instead supplied by free-
standing enzymes acting in-trans.[4] Such trans-AT PKSs
exhibit aberrant architecture and often incorporate novel
enzymatic domains, resulting in poor biosynthetic assign-
ments.[4b] Recent phylogenetic work established that KS
domains from trans-AT systems correlate, at the sequence
level, with their predicted biosynthetic intermediates. This
finding offers the exciting potential for KS-based PKS-to-
product assignment for trans-AT systems.[5]
The extent of evolution-based substrate correlation is
predicted to extend as far as the b-position for each
biosynthetic intermediate. Although the KS-specificity
method works remarkably well computationally, it lacks
functional testing. Such knowledge is important because it
could provide rules for biosynthetic engineering and may
allow for single-domain-based biosynthetic characterization
of trans-AT PKS megaenzymes. To address substrate specif-
icity in trans-AT PKSs, we have developed and applied a mass
spectrometry (MS)-based method to assign the specificity of
KS domains, thereby testing the predicted assignment of
trans-AT PKS clusters. Previous methods to determine the
specificity of the more familiar cis-AT PKSs have included
radioactivity assays and MS analysis of trypsin-digested
proteins.[6]
Herein, we report the substrate specificity of KS5 located
on BaeL of the bacillaene 1 PKS,[7] and KS1, KS2, KS30 on
PsyA/PsyD of the psymberin 2 PKS (Scheme 1).[8] Bacillaene
is an antibiotic isolated from Bacillus strains,[9] and psymberin
is produced by an uncultivated symbiont of a sponge.[10] Using
a rapid, MS-based method to analyze intact domains we show
that BaeL KS5 and PsyD KS30 do not accept an acyl chain
with methyl branching at the b-position, a common modifi-
cation of trans-AT PKS products.[4b,11] We rationalize this
specificity using homology modeling of the enzyme active
[*] M. Jenner, Dr. G. S. Briggs, Dr. N. J. Oldham
School of Chemistry, University of Nottingham
University Park, Nottingham NG7 2RD (UK)
E-mail: neil.oldham@nottingham.ac.uk
S. Frank, A. Kampa, C. Kohlhaas, P. Pçplau, Prof. Dr. J. Piel
Kekulꢀ Institute of Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
and
Institute of Microbiology,
Eidgençssische Technische Hochschule (ETH) Zurich
Wolfgang-Pauli-Strasse 10, 8083 Zurich (Switzerland)
E-mail: joern.piel@uni-bonn.de
jpiel@ethz.ch
Scheme 1. Partial proposed biosynthetic schemes for a) bacillaene
1 and b) psymberin 2. KS=ketosynthase; KS0 =non-elongating keto-
synthase; KR=ketoreductase; DH=dehydratase; CR=crotonase;
O-MT=O-methyl transferase; C=NRPS condensation domain;
[**] We are grateful for financial support by the Biotechnology and
Biological Science Research Council (BBSRC) and the University of
Nottingham to N.J.O. and the DFG (SFB 624 and PI 430/8-1) to J.P.
*
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201207690.
A=NRPS adenylation; GNAT=acetyl-loading AT; ACP domains ( ).
The sequential KS numbering is denoted above the KS domains, and
relevant carbon-branched regions of the intermediates are highlighted
in gray.
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sites, modulate the tolerance of BaeL KS5 with a single site-
directed mutation, and propose a general rule for the
tolerance of b-methyl-branching by KS domains.
40 minutes with SNAC derivative 6. This result provided
strong evidence that KS5 can not catalyze acylation by a b-
methyl-branched substrate. In contrast, the unbranched
SNACs 3–5 successfully acylated KS5, reaching saturation
after 20 minutes. O-branched SNACs were found to acylate
KS5, but at a slower rate than SNACS 3–5 (Table S2). MS
data obtained from incubation with SNAC derivative 5
resulted in additional signals that corresponded to the mass
of the attachment of the intact SNAC derivative (Figure S6).
This is explained by Michael addition to 5 by nucleophilic
sites on the protein surface. Conjugate addition did not
appear to interfere with the ability of KS5 to be acylated by 5,
and the two products were resolved by MS (something that
would not be possible with a radioactivity-based assay).
These results demonstrate that simple, short-chain SNAC
analogues of acyl-ACP intermediates are able to reveal
substrate specificity in KS5 of the bacillaene PKS. Notably,
this domain, which is positioned immediately before a b-
branching step, is highly specific for an unbranched substrate,
providing evidence that specificity studies of KS domains may
aid biosynthetic assignment.
The first three KS domains from the psymberin cluster
were then examined. Each of these KSs is predicted to use
different substrate intermediates (Scheme 1b). Substrate
profiling of PsyA KS1 revealed remarkably relaxed specific-
ity, with both branched and unbranched substrates accepted.
Kinetic analysis showed that the native acetyl substrate 3 was
readily incorporated (Table 1, Figure S7). However, the
butyryl derivative 4 was found to acylate at a comparable
rate, suggesting that chain length was not a dictating factor. In
contrast, the b-methyl-branched SNAC 6 was a worse sub-
strate, with rates approximately 30-fold slower than the
unbranched intermediates. However, unlike BaeL KS5,
carbon-branched SNACs were able to acylate PsyA KS1 to
some extent. Although initially hypothesized to be a highly
specific KS domain, owing to its position at the start of
a biosynthetic cluster and to its small substrate, functionally
KS1 is capable of accepting a range of substrates other than
the acetyl-SNAC 3. We postulate that there may be little or no
evolutionary pressure for KS1 to acquire specificity, as the
preceding GNAT domain supplies the correct acetyl starter
unit from malonyl-CoA.[13] The promiscuity of this KS
domain, together with its relatively high turnover, suggests
that it may be an attractive choice for biosynthetic engineer-
ing.
Previous sequence analysis places BaeL KS5 in a clade
with other KS domains predicted to accept an unbranched
a,b-unsaturated intermediate.[5a] KS5 is located before a key
b-branching step in the bacillaene biosynthetic pathway
(Scheme 1a), and would be expected to be specific for an
unbranched substrate.[11,12] To probe the specificity of this KS
domain, a range of simple N-acyl cysteamine (SNAC)
thioesters was synthesized (Table 1); these thioesters are
known to be suitable mimics of the acyl-phosphopantetheine
chain attached to the ACP.[6]
Table 1: Estimated initial acylation rates[a] for KS domains with SNAC-
thioesters 3–6.
Rate [ꢁ10À6 moldmÀ3 minÀ1
]
Substrate
BaeL
KS5
PsyA PsyA PsyD
BaeL KS5
(M237A)
KS1
KS2
KS30
3
4
5
6
0.07
3[b]
0.3
0.3
0.06
0.05
0.05
0.01
0.06
2
0.3
0.1
0.2
0.07[a]
2
0.3
ND
0.04
0.03
ND
[a] Initial rate was estimated from a plot of ln[KS]/[KS0] versus t, given to
one significant figure. [b] The SNAC-mimic of the predicted substrate
for each KS domain. ND=No acylation was detected over the time
scale of the incubation. Estimated error in measurements:
Æ0.005ꢁ10À6 moldmÀ3 sÀ1
.
Following incubation of KS5 with each SNAC derivative,
the reactions were quenched at set time points by addition of
0.1% TFA. After desalting (see Supporting Information),
electrospray ionization mass spectra of KS5 were obtained for
each time point. The mass of the non-acylated KS5 was
determined to be 78684 Da, in excellent agreement with the
predicted value of 78686 Da (Supporting Information, Fig-
ure S1). KS5 acylation was detected based on the appearance
of an additional signal corresponding to acyl-KS5 (Figure 1a).
Time-dependent acylation of the KS5 domain was measured
from the relative intensities of the non-acylated and acyl-KS5
peaks for each SNAC derivative and plotted (Figure 1b).
The E-2-butenoyl 5 and a 3-methyl-2-butenoyl-SNAC 6
thioesters were prepared as simplified analogues of the native
substrates of BaeL KS5 and KS6, respectively (Scheme 1 and
Table 1). MS-based kinetic analysis of KS5 with 5 and 6
revealed that this domain was highly selective for the
unbranched substrate, with no acylation observed after
Interestingly, PsyA KS2 produced a similar tolerance
profile for the SNAC-thioesters as KS1 (Figure S8). KS2 is
predicted to accept a b-branched intermediate, and 6 was
indeed found to be a viable substrate. KS2 was also acylated
by straight-chain acyl SNACs 3–5, as well as by O-branched
substrates (Table S2).
PsyD KS30 is a non-elongating KS, and is believed to
accept an (S)-b-hydroxyl intermediate (Scheme 1b). The
corresponding SNAC was found to be a viable substrate
(Figure S9, Table S2). The b-methyl-branched SNAC 6 was
not found to acylate KS30 after 40 minutes. Thus, with respect
to this substrate the specificities of BaeL KS5 and PsyD KS30
were rather similar, but different from those of PsyA KS1 and
KS2, for which tolerance for a b-carbon branch was seen.
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this domain appeared to display the highest levels of
specificity towards unbranched substrates. Using the CPHmo-
del server,[14] homology models of BaeL KS5 and the down-
stream, b-branch accepting, BaeL KS6 were successfully
constructed. Upon examination of the binding sites, a subtle,
but potentially important difference between KS5 and KS6
was observed. The nature of the residue immediately
preceding the active site cysteine (X-Cys) appeared to affect
the size of the binding pocket. X was identified as Met in
BaeL KS5 and Ala for KS6. To examine the effect of this
position on substrate binding, the unbranched biosynthetic
intermediate of KS5 was manually docked into the binding
site of KS5 (Figure 2a). Interestingly, the X residue was found
to be positioned adjacent to the b-position of the substrate,
with the potential to dictate the ability of the KS domain to
accept a b-branched substrate.
Applying this same method to the psymberin KS domains
revealed that PsyA KS1 and KS2 both had Ala residues at
position X, producing a binding pocket similar to BaeL KS6
(Figure S12). Interestingly, in this study, KS1 and KS2 have
been shown experimentally to accept the carbon branched
substrate 6. In contrast PsyD KS30 and BaeL KS5, which do
not accept this test substrate, possess a Met at the X position.
These results provide strong evidence for the role of a Met
residue in excluding bulky carbon b-branches. A sequence
analysis of the 138 KS domains used in the initial phylogenetic
study,[5a] together with 12 subsequently identified in the
psymberin PKS,[8] focused on the nature of the X-Cys
residues. Results from this analysis showed that for a large
proportion of known KS domains X = Ala. However, there
are some clades that deviate markedly from this norm. The
clade containing BaeL KS5, for example, comprises KS
domains that are believed to accept unbranched enoyl
substrates. Here, 94% of the KS domains in this clade feature
a Met residue at the X position (Table S3). This observation
added further evidence to the hypothesis that X = Met was
required to confer specificity towards unbranched substrates.
To test the hypothesis that the bulkier Met residue of
BaeL KS5 prevents acylation by carbon-branched substrates,
a BaeL KS5 (M237A) mutant was designed. The Met to Ala
mutation was predicted to provide the necessary space in the
binding pocket for a sterically demanding b-methyl branch
(see Figure 2B for homology model and branched substrate
docking). Incubation of the KS5 (M237A) mutant with the
unbranched SNACs 3–5 resulted in rates of acylation similar
to those of WT BaeL KS5. This was expected, as increasing
the space inside the binding cleft was unlikely to affect the
ability of an unbranched chain to acylate the KS. However,
there was a notable change in the tolerance towards the b-
branched test substrate. The carbon-branched SNAC deriv-
ative 6 was found to acylate BaeL KS5(M237A) at a reason-
able rate, demonstrating that the mutation had altered the
specificity of the KS domain. These observations suggest that
KS domains from trans-AT PKSs have the ability to differ-
entiate substrates by the extent of b-branching, but can be
engineered to accept non-natural acyl units.
Figure 1. Acylation of BaeL KS5 using SNAC thioesters. a) Stacked
mass spectrum of the 59+ charge state of BaeL KS5 showing acylation
by SNAC 4. Kinetic plots of b) WT BaeL KS5 and c) BaeL KS5(M237A)
with SNAC thioesters 3–6.
A combination of homology modeling and sequence
analysis was employed to rationalize the observed differences
in KS specificity. Initial focus was directed on BaeL KS5, as
Although the use of short SNAC thioesters has allowed
elucidation of a certain level of specificity in the KS domains
we studied, the specificity is notably lower than that predicted
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Figure 2. Homology models of KS domains. a) WT BaeL KS5 with a predicted, unbranched biosynthetic intermediate docked. The methionine
residue highlighted in red is postulated to provide the necessary steric bulk to prevent b-branched substrates entering the binding pocket. b) BaeL
KS5 (M237A) with the b-methyl analogue docked. In this case, the alanine residue highlighted in blue is thought to reduce the steric crowding in
the binding pocket, allowing a b-branched substrate to bind. PPant represents the phosphopantetheinyl chain of the preceding ACP domain.
by phylogenetic analysis.[5a] This may reflect low specificity at
the functional level, or might be an artifact from the use of
simplified substrates. Extension of the acyl chain to better
mimic that of the natural substrate, or use of the full
pantetheinyl chain, instead of the simple SNAC thioester,
may allow higher-resolution insight into KS specificity.
The current hypothesis concerning the evolutionary origin
of cis-AT and trans-AT PKSs is that they have evolved by
different routes.[5a] It has been suggested that the KS domains
from trans-AT PKSs developed through horizontal gene
transfer, by assembly of substrate-specific KS domains.[5a,15]
This is in contrast to cis-AT KS domains, which have evolved
as a result of duplication of entire modules in which KS
domains are embedded.[16] A prerequisite for this evolu-
tionary-duplication mode is relatively low substrate specific-
ity, which is indeed observed for many cis-AT KSs.[17]
Examination of the X-Cys active site motif in KS domains
from cis- and trans-AT PKSs revealed interesting differences
between the two types (Figure S13). For trans-AT PKSs,
which accept a- and b-carbon-branched substrates, X is
always a less sterically demanding residue Ala or Gly. Also, X
is exclusively Asn for KSs that use amide-containing sub-
strates. cis-AT PKSs, in contrast, might not be expected to
have evolved the variety of amino acids at the crucial X
position to develop this mechanism of specificity. Examina-
tion of the available sequence data reveals that X is Ala for all
cases of fully assigned cis-AT PKSs in the literature (Fig-
ure S13). This observation adds further evidence to the theory
that different modes of evolution have occurred in cis and
trans-AT PKSs.
favorable cases; specifically in highlighting the incompatibil-
ity of proposed b-methyl intermediates with X = Met KS
domains. Additionally, these results indicate the potential to
tune the specificity of KS domains towards desired biosyn-
thetic products.
Received: September 24, 2012
Published online: December 5, 2012
Keywords: biosynthesis · electrospray ionization ·
.
ketosynthases · mass spectrometry · polyketides
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