Báo cáo khoa học: The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry pptx

The changing patterns of covalent active site occupancy
during catalysis on a modular polyketide synthase
multienzyme revealed by ion-trap mass spectrometry
Hui Hong1,2, Peter F. Leadlay2 and James Staunton1
1 Department of Chemistry, University of Cambridge, UK
2 Department of Biochemistry, University of Cambridge, UK

Keywords
enzyme-bound intermediate; erythromycin;
limited proteolysis; liquid chromatographymass spectrometry; polyketide synthase
Correspondence
H. Hong, Department of Biochemistry,
University of Cambridge, 80 Tennis Court
Road, Cambridge CB2 1GA, UK
*Fax: +44 1223 766002
Tel: +44 1223 333659
†E-mail:
J. Staunton, Department of Chemistry,
University of Cambridge, Lensfield Road,
Cambridge CB2 1EW, UK
Fax: +44 1223 336362
Tel: +44 1223 336300
E-mail:
(Received 28 July 2009, revised
29 September 2009, accepted 30
September 2009)
doi:10.1111/j.1742-4658.2009.07418.x

A catalytically competent, homodimeric diketide synthase comprising the
first extension module of the erythromycin polyketide synthase was analysed
using MS, after limited proteolysis to release functional domains, to determine the pattern of covalent attachment of substrates and intermediates to

active sites during catalysis. Using the natural substrates, the acyltransferase
and acylcarrier protein of the loading module were found to be heavily
loaded with propionyl starter groups, while the ketosynthase was fully propionylated. The acylcarrier protein of the extension module was partly occupied by the product diketide, and the adjacent chain-releasing thioesterase
domain was vacant, implying that the rate- limiting step is transfer of the
diketide from the acylcarrier protein to the thioesterase domain. The data
suggest an attractive model for preventing iterative chain extension by efficient repriming of the ketosynthase domain after condensation. Use of the
alternative starter unit valeryl-CoA produced an altered pattern, in which a
significant proportion of the extension acylcarrier protein was loaded with
methylmalonate, not diketide, consistent with the condensation step having
become an additional slow step. Strikingly, when NADPH was omitted, the
extension acylcarrier protein contained methylmalonate and none of the
expected keto diketide, in contrast to results obtained previously by mixing
individual recombinant domains, showing the importance of also studying
intact modules. The detailed patterns of loading of the extension acylcarrier
protein (of which there are two in the homodimer) also provided the first
evidence for simultaneous loading of both acylcarrier proteins and for the
coordination of timing between the two active centres for chain extension.

Introduction
Polyketides are a large and diverse group of secondary
metabolites that are produced by a common biosynthetic strategy in bacteria, fungi, plants and animals.

The term polyketide refers to the early steps of a typical
pathway, in which a starter acyl residue is extended by
successive addition of acyl residues, each equivalent to

Abbreviations
ACP, acyl carrier protein; AT, acyl transferase; DEBS, 6-deoxyerythronolide B synthase; DKS, diketide synthase; FAS, fatty acid synthase;
KR, ketoreductase; KS, ketosynthase; PKS, polyketide synthase; SNAC, thioester of N-acetylcysteamine; TE, thioesterase.
* [Correction added on 6 November 2009 after first online publication: The fax number is wrong, it should be 766002, not 966002].

† [Correction added on 6 November 2009 after first online publication: the email address for the first corresponding author is wrong, it
should be , not ].

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H. Hong et al.

the general structural ketene unit, RCH=C=O, until
the linear chain of carbons reaches the desired length.
Subsequent diverse biosynthetic transformations generate an enormously varied set of structures [1,2].
The catalytic enzymes responsible for the chain-extension processes show a remarkable degree of structural
and mechanistic homology across the wide range of biosynthetic organisms. There are large differences, however, in the manner in which the enzymes are housed in
multienzyme clusters. At one extreme, a single set of
chain-extension enzymes carries out all the chain-extension steps (iterative operation); at the other extreme,
there are systems which have a separate enzyme for
every step of the chain-extension processes (modular
assembly line operation). A further source of variance is
found in the nature of association between enzymes in
the clusters; in some systems (Type II) the individual
enzymes are readily dissociable; others (Type I) contain
large assemblies of covalently linked catalytic sites.
The work in this investigation applies exclusively to
Type I modular systems that occur largely in bacteria
and produce so-called ‘complex’ polyketides. These
polyketides are a very large and diverse group of secLoad

Module 1

Module 2

Module 3

DEBS1

ER

DEBS2

SH

S

ACP KS AT ACP TE
SH

SH

S

S

O

O

O

OH

OH

OH

OH
S

S

O

OH

O

KS AT

SH

S

S

KR

KR

KS AT ACP KS AT ACP

SH

SH

Module 6
DEBS3

KR

DH

AT ACP KS AT ACP KS AT ACP

Module 5

Module 4

KR

KR

ondary metabolites, produced largely (if not exclusively) by certain genera of bacteria. They include
some of the most valuable natural products to have
reached the clinic, such as the antibacterial erythromycin A, the antiparasitic avermectin and the immunosuppressant rapamycin [1]. They are produced on giant
modular multienzyme assembly lines [polyketide synthases (PKSs)] in a linear chain-building process akin to
that of fatty acid biosynthesis, with the chief difference
being that PKSs may recruit a far greater variety of

starter units and extender units, while the overall
length of the product and the level of reduction of
each unit, as it is incorporated, may also vary [2–5].
The specificity is assured by utilizing a different module of fatty acid synthase (FAS)-related activities for
each cycle of chain extension, as illustrated in Fig. 1
for the erythromycin-producing PKS [6-deoxyerythronolide B synthase (DEBS)], which catalyzes assembly of
the aglycone 6-deoxyerythronolide B from one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA [2,5–8]. Figure 1 also shows the
arrangement of enzymatic domains in an engineered
diketide synthase (DKS) containing only one extension

O

O

O

OH

OH

O

Me
Me
O

OH

Cyclise on
TE domain

OH

O

OH

OH

O

OH

OH

Me
Me
Load

OH

OH

Me

Intermediates

Module 1

Me

O
O

OH
OH

6-Deoxyerythronolide B
(6-DEB)

Me
DKS

OH

KR

Me

AT ACP KS AT ACP TE
OH

OH

SH OH
S

S

Hydrolyse

on TE
domain

HO
O

O

O

OH

Me

OH
Me

Me
Diketide acid

Fig. 1. PKS assembly line responsible for assembling the macrolide core of 6-deoxyerythronolide B (6-DEB), as revealed by sequencing the
genes. Each cycle of chain assembly is carried out by a dedicated set of enzymes so that there is a separate enzyme for every step. The
enzymes are organized in sets (modules), one for each cycle. Each module has the correct set of enzymes for the extent of keto group
modification (hydroxyl, enoyl, saturated methylene, colour coded), which ensures that the fatty acid chain-extension cycle is appropriately
truncated and the correct transfer path is followed. At the terminus there is a TE domain that carries out cyclisation and release of the first
enzyme-free product, 6-DEB, as a macrolactone. The truncated model DKS is shown below. It carries out the operations of module 1 and
then releases the diketide product as a fatty acid.

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H. Hong et al.

module [9], from which the diketide product is released
by the action of the C-terminal thioesterase (TE). The
purified DKS, generated from DEBS by deleting extension modules 2–6, has been shown to hydrolyze and
release the predicted diketide product in vitro [9], albeit
with a turnover that is low compared with the betterstudied triketide synthase, DEBS1-TE, where the TE
can operate its preferred mode of chain release via
cyclization [10].
At first sight, the modular paradigm for enzymatic
catalysis in these modular PKSs, one of which is shared
with nonribosomal peptide synthetases [11], appears
simple because of the apparently direct correspondence
between the enzyme activities of a given module and
the chemical structure produced during that cycle of
extension. However, despite extensive study, it is still
not understood how these giant enzymatic assemblies
are controlled and orchestrated. In fact, under certain
conditions, modular PKSs have been shown to give
aberrant products in vivo in which individual enzymecatalyzed steps [12], or even whole modules, are
‘skipped’ [13,14], while in other cases extension modules operate more than once (iteration or ‘stuttering’)
[15–17]. In a few examples, such skipping or iteration is
actually required in order to produce the natural product [18–21]. Recent structural studies on the intact animal FAS multienzyme [22,23] and on modular PKS
domains [24–27] have also given a fresh impetus to the
question of control and orchestration of the individual
steps involved in chain extension. The work on FAS
has revealed a high mobility of certain domains and

potentially a key role for major conformational
changes during catalysis [22,23]. Both animal FAS and
modular PKS are functional homodimers, which raises
additional questions about the interactions between the
active sites of an identical pair of modules.
We report here the use of ion-trap MS and the DKS
model system [9] to study the identity of multienzymebound intermediates and to establish the pattern and
level of covalent attachment of substrates and intermediates to individual active sites, during catalysis in vitro
on a modular PKS. For dissociated (Type II) FAS and
PKS, monitoring the nature of acyl chains attached to
the acylcarrier protein (ACP) followed quickly upon
the introduction of electrospray MS [28,29] and it
continues to give valuable mechanistic insights [30,31]
into such systems.
Unfortunately the sheer size of modular PKS multienzymes has hampered their analysis in this way. One
successful approach [32,33] was to degrade the multienzymes to short peptide fragments, fractionate the
complex mixture and use high-resolution electrospray ionization Fourier-Transform mass spectrometry

Changing patterns of covalent active-site occupancy

(ESI ⁄ FTMS) to pick out the active-site peptides and
determine the nature of the covalently attached group
[34,35]. In addition, specific ejection of the phosphopantetheinyl prosthetic group from the ACP can be
induced in the mass spectrometer, allowing highly accurate determination of the mass of the attached species
[35]. A convenient ion-trap MS-based approach may
also be used in which limited proteolysis [36] is used to
generate domain-sized fragments for liquid chromatography (LC) ⁄ MS analysis. We have previously validated
this technology for the DKS (Fig. 2A) [37] and used it
to demonstrate, for the first time, that during the loading of the synthase with the natural starter unit propionyl-CoA (as well as from the alternative starter units
acetyl-CoA, butyryl-CoA and valeryl-CoA), three different active sites in both DKS subunits become almost

completely acylated, namely the acyltransferase (AT)
and ACP domains of the loading module and the ketosynthase domain (KS1) (Fig. 2B). Here, we have used
the method to establish the pattern of covalent intermediates attached to various active sites of an intact PKS
module, during catalysis of overall diketide formation.
This has revealed previously unsuspected features of
chain elongation on such enzymes.

Results
The same methodology as used in our previous study
[37] was first applied to investigate the loading of the
natural methylmalonyl extender unit, derived from
methylmalonyl-CoA, onto the extension module ACP
(ACP1) of DKS. After incubation with commercial
methylmalonyl-CoA for 10 min, the DKS protein was
digested and subjected to analysis by HPLC ⁄ MS. The
extension module AT domain from DKS (AT1)
appears as two fragments corresponding to alternative
sites of proteolysis; one fragment has a molecular mass
of 32582 Da and the second has a molecular mass of
32739 Da. As expected, after incubation with methylmalonyl-CoA, both fragments showed two extra peaks
at 32684 and 32839 Da, respectively, corresponding to
the addition of a methylmalonyl moiety (see Fig. 3).
The ratios of the intensities of the peaks for the loaded
and unloaded forms of each AT1 fragment were
almost identical, at 60% and 40%, respectively
(Table 1). Similarly, the fragment for the ACP-TE
di-domain showed two peaks, one with a molecular
mass of 39506 Da corresponding to the unloaded form
and the other with a molecular mass of 39604 Da, corresponding to the form loaded with methylmalonate
(see Fig. 3). In this case, a ratio of approximately 55%

loaded to 45% unloaded forms was observed
(Table 1).

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A Control experiment; no incubation prior to analysis

Load

Module 1
KR

Proteolysis
AT ACP

AT ACP KS AT ACP TE
OH

SH OH

OH

AT

KR

ACP TE

OH

SH

OH

SH

SH

KS

OH

TE
OH

SH

SH

B After incubation with propionyl CoA

Load

Module 1
KR

Proteolysis

AT ACP KS AT ACP TE
O

S
O

OH
O

AT ACP

OH

KS

O

S

KR

ACP TE
OH

OH

S
O

SH

AT
O

TE
OH

SH

S
O

O

C After incubation with purified methylmalonyl CoA

Load

Module 1
KR

Proteolysis

AT ACP KS AT ACP TE
OH

SH O
SH O
HO2C

OH

AT ACP

SH

OH
SH

S
O

KS

KR

AT

ACP TE
OH

O
O
CO2H

TE
OH

S
O
CO2H

CO2H

Fig. 2. Results of proteolysis of the DKS followed by HPLC coupled to electrospray MS. Experiments (A) and (B) were reported in a previous publication [37]; experiment (C) is part of the current results.

Surprisingly, analysis of the fragments derived from
the loading module in this initial experiment produced evidence that both the AT and ACP domains
of the loading module, and the KS domain, were
loaded with propionate. The purity of the commercial
methylmalonate was therefore checked by HPLC,
which revealed contamination with propionyl-CoA.
Although the level of contamination was low, it was
sufficient to explain the unexpected propionate loading. Purification by HPLC gave propionyl-CoA free
of the methylmalonyl analogue. Repeating the experiment gave the same results for loading of AT1 and
ACP-TE di-domains, but the domains of the loading
7060

module and the KS were completely unloaded, as
indicated in Fig. 2C. Apart from demonstrating the
importance of using pure materials, the results with
the pure methylmalonate were significant in removing
any possibility that methylmalonate decarboxylation
by the KS provides an alternative source of the propionate building block used in the first condensation
step. The DKS was also incubated with malonyl-CoA

under the same conditions used for methylmalonylCoA. No loaded residues were detected for any of
the domains, showing that the AT1 domain is highly
substrate specific, unlike the AT domain of the loading module (data not shown).

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H. Hong et al.

Changing patterns of covalent active-site occupancy

AT

ACP-TE
Methylmalonyl
adduct
Methylmalonyl
adducts

Unloaded
forms

Fig. 3. The electrospray mass spectra produced by the AT and ACP-TE fractions
derived from the extension module 1 after
incubation with methylmalonyl-CoA. The AT
domain shows two sets of peaks that arise
from alternative sites of proteolysis in the
downstream linker to KR.

32 400

32 800

Unloaded
form

33 200

39 400

39 500 39 600

Mass (Da)

The DKS was then incubated with various combinations of substrates that should allow synthesis of a
diketide product and then analyzed as before to determine the nature and extent of occupancy of the KS,
AT and ACP chain-extension domains of module 1
[the ketoreductase (KR) domain does not have a substrate covalently bound] and of the adjacent TE
domain. Various sets of incubation conditions, listed
in Table 1, were explored. The possible adducts on the
various domains are shown in Fig. 4. First, the DKS
was incubated with propionyl-CoA to supply the
native starter unit, methylmalonyl-CoA as the source
of extender unit, and, with NADPH, to carry out the
keto-group reduction step catalysed by the KR
domain. After a sufficient incubation period to establish steady turnover (10 min), the mixture was analysed
using the standard protocol to determine the extent of
loading on the domains of module 1 and its attached
TE domain.
MS analysis of the KS1 fraction showed that the

active site was fully loaded with propionate. The
absence of the free form of the KS shows that loading
of propionate onto the KS via the loading module is

39 700

Mass (Da)

not rate-limiting under these conditions. The mass
spectrum for the fraction containing the TE domain
and the key ACP-TE domain is shown in Fig. 5B.
Only one peak was observed for the TE domain, with
a mass corresponding to the unloaded form. From this
it can be concluded that release of the diketide intermediate from this domain is faster than its acylation
by diketide transfer from ACP1. Therefore any covalently attached species detected on the ACP-TE
di-domain is resident only on the ACP1 thiol. Two
peaks are seen, one corresponding to the unloaded
form and the other to the form loaded with diketide
(see Table 1). From the increased mass this could have
been the keto diketide form or the hydroxy diketide
form, or a mixture of the two. Surprisingly, given the
results of incubation with methylmalonyl-CoA alone,
there was no evidence for the methylmalonyl derivative
of the chain-extension ACP, despite the presence of
the free thiol form of the domain.
The nature of the diketide adduct in this experiment
was determined by treatment of a sample of the loaded
ACP-TE di-domain with hydrazine to remove the
added diketide ligand as the hydrazide derivative.

Table 1. Occupancy levels of intermediates on chain extension ACP1 under various assay conditions.
Percentages of derivatized forms of the chain-extension ACP domain
Incubation mixtures

Free thiol

Methylmalonate

Ketodiketide

Hydroxydiketide

1.
2.
3.
4.
5.
6.

45
0
42
51
37
45

55
0
0
0

35
55

N⁄A
N⁄A
0
0a
0a
0

N⁄A
N⁄A
58
49a
28a
0

a

Methylmalonyl-CoA
Malonyl-CoA
Propionyl-CoA; methylmalonyl-CoA; NADPH
Butyryl-CoA; methylmalonyl-CoA; NADPH
Valeryl–CoA; methylmalonyl-CoA; NADPH
Propionyl-CoA; methylmalonyl-CoA; no NADPH

Absence of keto-diketide assumed by analogy with experiment 3.

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A

KR
AT

KS

ACP

AT

OH

SH

SH

OH

O

S

S

TE

ACP

OH

S

SH

B
O
R

O
R

O

O

O

O

R
CO 2H

OH
R

C
S

S
O

CO 2H

S
O

O

O
R

OH
R
28 000

Fig. 4. The range of biosynthetic intermediates predicted to be
covalently bound to the various enzyme active sites in the course
of a chain-extension cycle and product release on the DKS.

32 000

36 000

40 000

Mass (Da)

D

Free
39 509
Methylmalonate
39 611

First, suitable conditions for the reaction were established by a control study with a synthetic sample of
the N-acetylcysteamine analogue (Fig. 6). The extract
of the reaction mixture showed a product which was
identified by high-resolution MS (calculated for
expected product [M+H]+ 147.1133, found [M+H]+
147.1150). Further support for the structure of the
hydrazide was obtained from an MS ⁄ MS spectrum,
which produced a fragment ion for loss of H2O at m ⁄ z
129.1. Repeating this experiment with the loaded ACPTE di-domain gave the same product, as judged by
MS analysis. Careful examination of the LC-MS trace
failed to show evidence for any of the possible hydrazine derivatives of the keto analogue of the diketide
(m ⁄ z 145 or 127), and so a confirmatory control experiment with the keto derivative was not necessary.
Analysis of the ACP-TE fraction recovered from the
hydrazine treatment showed it to be in the free form,
as expected. These experiments confirmed that the
DKS is active in diketide synthesis under these conditions, and that the hydroxy diketide intermediate accumulates on the synthase in a significant quantity. It
appears that very little, if any, of the accumulated
diketide intermediate is in the unreduced keto-form.

When the natural starter unit was replaced with
butyrate (an increase in size of 14 mass units), analysis
of the DKS showed that the KS domain was fully
loaded by butyrate. As with propionate, the chainextension ACP (ACP1) showed a peak for the
unloaded form and a peak for the form loaded with
the heavier diketide analogue (Fig. 5C). The two
7062

Diketide
39 644

39 300

39 500

39 700

39 900

Mass (Da)

Fig. 5. MS results from experiments 3, 4 and 5. (A) Control experiment without added precursors. (B) Incubation with propionate as
the starter in experiment 3. (C) Incubation with butyrate as the starter in experiment 4. (D) Incubation with valerate as the starter in
experiment 5, showing the expanded version of the ACP-TE region.

species were now present in approximately equal
amounts (Table 1). Again, the isolated TE domain was
free of diketide derivative, and there was no evidence
for the methylmalonyl derivative of the ACP. Incubation of DKS with valeryl-CoA likewise led to the complete loading of KS with the valeryl group, but here
there was a marked change in the pattern of loading

of ACP1. This now showed three peaks: free thiol group
(37%), the valeryl diketide derivative (28%) and, in
addition, the methylmalonyl derivative (35%) (Fig. 5D)
not seen when either propionyl-CoA or butyryl-CoA
was used (Table 1). As in the previous two experiments,
the TE was not found to be acylated.

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H. Hong et al.

Changing patterns of covalent active-site occupancy

ACP TE
OH
SNAC
O

H2NHN
NH2NH2

OH

O
OH

NH2NH2

S

hydrolase activity [38], which may also influence the
steady-state level of acylation of AT1 and ACP1
domains.

O
OH

Fig. 6. Chemical treatment of the diketide N-acetylcysteamine
(NAC) derivative and the ACP-TE derivative with hydrazine to
release the hydrazide product for analysis by MS. SNAC, thioester
of N-acetylcysteamine.

Finally, an experiment was carried out in which the
natural substrates propionyl-CoA and methylmalonylCoA were supplied, but in which NADPH was not
supplied. The aim was to see if the keto-ester intermediate accumulated, and, if so, which stereoisomer dominated in the keto-ester product. As expected, the KS
domain was found to be fully loaded with a propionyl
unit, and the two domains of the loading module were
also substantially loaded with the starter acyl residue
units. However, the omission of NADPH had a
marked effect on the pattern of loading on the chainextension ACP (ACP1) in this experiment. There was a
peak for the methylmalonyl derivative, as well as for
the free thiol form, but there was no detectable peak
for any diketide intermediate. Interestingly, the relative
proportions of the free thiol form of ACP1 (45%) and
the methylmalonyl form (55%) were identical to those
observed in the experiment in which no starter unit
was supplied, and the TE domain was again unloaded.

Discussion

Incubation of DKS with methylmalonyl-CoA gives
incomplete acylation of AT1 and ACP1 domains
In our previous experiments [37] we showed that
incubation of the DKS in vitro with saturating concentrations of starter substrates led to complete acylation of the KS domain and nearly complete acylation
of both domains in the loading module. By contrast,
in the present study we found that the level of loading of methylmalonate on both the chain-extension
AT and ACP domains was considerably less than
100%. As the AT-catalyzed reaction is readily reversible, the extent of loading of methylmalonate on the
AT and ACP domains is probably determined (at
least in part) by the relative stabilities of free methylmalonyl-CoA ester and the loaded forms of the
domains, and thus by the concentrations of methylmalonyl-CoA and protein used in this study. We have
also previously demonstrated that the AT domains of
purified DEBS possess a slow methylmalonyl-CoA

Identification of rate-limiting steps, and a model
for suppression of iteration and the maintenance
of fidelity of reduction
In the presence of all the (natural) substrates required
for diketide synthesis on the DKS, the relatively high
level of loading of multiple sites (> 50%) persists,
apart from the TE domain. It would appear that under
these conditions, two chains can be elongated at the
same time, and that the rate-limiting step is the transfer of the diketide intermediate from the chain-extension ACP to the TE, not the subsequent release of the
diketide acid from the TE. This bottleneck at the exit
stage causes a backlog of intermediates to build up at
previous steps.
When the alternative (progressively poorer) starter
substrates butyryl-CoA and valeryl-CoA were used,
the KS remained fully loaded and the extent of
methylmalonate loading on the chain-extension AT

was not significantly changed. By contrast, there
were dramatic changes in ACP1 occupancy. With
butyrate, the proportion of ACP1 loaded with diketide fell significantly, consistent with a slowing of the
rate of the condensation step relative to the offloading step (we assumed that all the diketide intermediate was in the hydroxy form in experiments 4 and
5). In the valerate experiment there was a more dramatic change. The proportion of the diketide intermediate fell even further (below 50%) and a
detectable amount of ACP1 was found to be loaded
with methylmalonate. It would appear that the condensation step, under these conditions, provides a
significant additional bottleneck in the chain-assembly process. However, the key point to emerge from
these experiments is that by using the normal substrates, all the active sites are found to be heavily
loaded. This situation, if it holds for PKSs in vivo,
would contrast sharply with a conventional metabolic
pathway where overall rate control is dominated by
early enzymes to avoid the accumulation of large
pools of enzyme-free intermediates.
Regulation of productivity at the release step could
also provide important advantages for the control of
fidelity in natural modular PKS assembly lines. In the
case of the complete DEBS assembly line, for example,
regulation at the stage of product release would cause
all the ACP and KS domains to be loaded by appropriate intermediates and all the AT domains to be
primed with the relevant building blocks. This would

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lead to an intermittent mode of operation, in which
successive rounds of chain-extension cycles would be
triggered by release of the heptaketide intermediate
from the ACP domain in the last module (Fig. 1),
rather like the operation of an automatic drinks-can
dispenser. In each module, when the KS domain has
become free, it would be immediately reloaded with
substrate from the upstream ACP. This would prevent
back-transfer of biosynthetic intermediates subsequently generated on the ACP domain within that
module, and suppress iterative use of the module. The
term ‘congestion control’ has been suggested for this
effect [5]. It is consistent with this hypothesis that
aberrant iteration in a PKS has been seen when the
levels of a PKS were increased without also increasing
the levels of intracellular precursors [15].
The throttling back of the process of product
release could also contribute to the maintenance of
fidelity in the reductive steps of the DEBS synthetic
operations. It is vital that in every cycle all the programmed reductive steps are completed before downstream transfer of the fully modified intermediate
from the ACP to the KS domain of the next module.
Molecular recognition cannot be the sole factor in
this, as shown most clearly for the mycolactone PKS,
where all 16 extension KS domains have an essentially identical sequence [39] and cannot therefore be
expected to discriminate between the various intermediates sequentially generated in the upstream module.
An alternative and simpler mechanism for the control
of fidelity is that all the steps of keto group modification go effectively to completion, under the conditions
in which PKSs operate; and that in every module the
downstream transfer of product to the KS of the next
module is delayed because it remains loaded until the

modification reactions have had time to reach completion. The term ‘retardation control’ has been suggested for this proposed effect [5]. The implied high
level of loading in all the modules of modular PKS
multienzymes is consistent with the suggested ‘leaky
hose’ explanation for the release of biosynthetic intermediates from the mupirocin PKS when downstream
catalytic sites are blocked [40].
Maintenance of fidelity in the reductive steps relies
not just on suppression of iteration, and of premature
transfer of incompletely reduced polyketide chains to
the next extension module, but also on precise control
of reaction stereospecificity by way of molecular recognition between substrates and individual ketoreductase,
dehydratase and enoylreductase domains [10,41–44].
Perturbation of these interactions leads to inactivation,
or to the generation of aberrant products, both in vivo
[12,42] and in vitro [43].
7064

Evidence for coordination of condensation and
ketoreduction
In previous work [44] the operation of a single extension module from DEBS was studied in vitro by mixing individually expressed and purified domains (ACP,
KR) and di-domains (KS-AT). This flexible approach
allowed various combinations of each type of domain
to be assayed and easily analyzed, and for individual
steps to be deconvoluted. For example, when a KS-AT
di-domain was incubated with a diketide thioester
substrate, methylmalonyl-CoA and ACP, keto triketide
attached to the ACP was efficiently formed. We therefore expected that when DKS was incubated with
substrates in the absence of NADPH, the ACP1 would
be found to carry the keto diketide. However, under
the conditions used, we found only the building blocks
loaded on the KS and ACP1 domains respectively, and

no diketide intermediate. Given the surprising nature
of this result, the experiment was repeated many times,
always with the same outcome.
It appears that either the keto diketide intermediate
is subject to very rapid release by the TE, or the condensation step is seriously inhibited in the absence of
NADPH. The former explanation can be ruled out
because no evidence for a ketoacid by-product was
found in any of our careful searches for by-products in
early investigations of the diketide synthase in vivo or
in vitro [9,45]. The possible existence of an unexpected
allosteric effect that inhibits the condensation step
therefore deserves consideration. The missing ingredient, NADPH, would be expected to bind to the KR
domain, not to either of the domains involved in the
condensation step. Any effect is therefore remote and
must depend on the quaternary structure. A similar
inhibition of loading of methylmalonate onto an ACP
was also reported in a study of the epothilone synthase, and again the effect was attributed to the quaternary structure [33]. Figure 7 shows a schematic
representation of the arrangement of domains within
the extension module of the DKS, based originally on
modelling and detailed proteolytic studies of DEBS
that established the homodimeric nature of PKSs [46],
and incorporating subsequent evidence from functional complementation [47], NMR studies [26] and
X-ray crystal structures of intact animal FAS and of
DEBS domains and di-domains [8,22]. There is now a
consensus that the two polypeptide chains in a homodimeric PKS are aligned ‘tail to tail’, as well as
‘head to head’, in each module, as predicted [46]. In
the absence of a crystal structure for an intact PKS
module, it remains unclear how the two KS domains
and the two ACP domains are able to approach

FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS


H. Hong et al.

Changing patterns of covalent active-site occupancy

AT1

KR1

KS1

ACP

KS1

ACP

AT1

TE

TE

KR1

closely enough to co-operate in the condensation, but
one suggestion is shown in Fig. 7. The two ACP
domains move, perhaps in unison, along the axis

through a central passage, and so make contact with
the upstream pair of KS domains. The double-helical,
rope-like twist of the two chains provides evidence
that the ACP of one chain makes contact with the
KS of the other [46,47]. The cartoon shown here is
equivalent to the earlier ribbon representation [46],
except that the two-fold axis of symmetry runs horizontally rather than vertically, and the alternative
direction of the helical twist is adopted in accordance
with that established from the recent X-ray structure
produced for the KS-AT di-domain [24].
In this working model of the DKS, the AT and KR
domains form a ‘collar’ surrounding the backwards and
forwards path of travel of the two ACP domains, as
they interact with their various catalytic partners. The
collar shelters the central region and protects the biosynthetic intermediates from the surrounding aqueous
medium. If the collar can expand and contract to control the lateral passage of the ACP domains, there
exists a possible mechanism by which the presence of
NADPH might enable the condensation step by binding to the KR and inducing a conformational change
that opens the collar. The AT domain may also be
involved in such movements, as although the KS-AT
linker exists with a tightly folded tertiary structure in
the X-ray structure of the di-domain [24], this interdomain region is readily cleaved by the mild conditions of
proteolysis conditions used in the present study. It is
tempting to speculate that the absence of methylmalonyl loading of ACP1 when a readily converted substrate
(propionyl-CoA) was used, compared with the significant level of methylmalonyl loading of ACP1 with a

Fig. 7. Proposed quaternary structure of the DKS chain-extension
module based on the topology of the Cambridge Double Helical
Model. The two identical chains of the homodimeric structure are
differentiated by red and blue colouring. The KS domains have

strong homodimeric interfaces (purple blocks) and are placed in
contact at the ‘head’ of the structure. The strongly homodimeric
TE domains are also placed in contact with each other at the
‘tail’. The remaining three domains are not homodimeric and so
can move away from the common axis running through the pairs
of KS and TE domains. The pair of ACP domains, however, are
held close to the TE domains by short linker regions and so must
remain in close proximity to each other and to the axis. To aid
visualization, the two domains, KR and AT, in the mid-section of
the homodimer, are shown as small black blobs rather than as
coloured spheres of appropriate size. These domains are sited
away from the axis of the proposed structure to free up a central
passage. The pair of ACP domains can now make contact with
the pair of KS domains by moving parallel to the axis with the TE
domains in tow. The structure is also given a helical twist of 180
degrees in accordance with evidence that the ACP of one chain
interacts with the KS of the opposite chain. In the resulting quaternary structure, each ACP domain can access the appropriate
KS domain for the condensation step (and other domains in succession through the chain extension cycle) by moving backwards
and forwards (dashed green arrows) along the axis within the central core of the structure. Because of the restricting effect of the
short ACP to the TE linker, and the anticipated need for co-ordinated movements of the two looped-out domains, it is likely that
the pair of ACP domains move backwards and forwards in tandem, rather than independently. As a result, the successive reactions of the chain-extension cycle on the two chains might also
be constrained to operate in tandem. At the start of each step of
the chain-extension cycle, the pair of ACP domains would be
loaded with identical intermediates and both would bind with the
appropriate domain for the next step. The first intermediate to
complete the reaction would be free to leave its catalytic domain,
but its ACP would stay put until the second intermediate had
completed the same operation. Both intermediates are now free
of the catalytic sites and the two ACP domains would then move
in tandem to co-operate with the next pair of catalytic domains.

In the Figure, the relative positioning of the KS and AT domains
conforms to that shown in the X-ray structure of an isolated KSAT fragment [24] with the AT domains in an outer position,
remote from the axis. However, the KS–AT linker undergoes facile
proteolysis, so it must be in equilibrium with an unfolded form,
not revealed in the X-ray image. With the linker unfolded, the two
AT domains would be free to move to inner positions closer to
the axis. In the outer position, they would reload with methylmalonate. The subsequent move to an inner position (the original
cartoon illustrating the structural principles of the Double Helical
topology showed this quaternary conformation [46]) would facilitate transfer of the methylmalonate to the ACP, and, in addition,
would block premature access of the unloaded ACP thiol to the
KS active site, a situation that would lead to skipping of the module. These predictions of co-ordinated movements of domains in
the Cambridge Double Helical Model are consistent with the
intriguing patterns of occupancy of ACP1 that are revealed in this
investigation.

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Changing patterns of covalent active-site occupancy

H. Hong et al.

less readily converted substrate (valeryl-CoA), is preliminary evidence of a threshold level (50%) of diketide
attachment to one of a pair of ACP1 domains being
sufficient to suppress the movement of the unloaded
partner ACP1 towards the AT1 domain for re-acylation
to occur. A requirement for the ACP domains to
move in tandem could neatly account for such effects.

Unfortunately, in cutting up a homodimeric megasynthase to facilitate MS analysis, a crucial aspect of
the loading information is destroyed: the analysis
reveals the average level of loading across all the individual domains in the homodimeric species, but, in
modules that are only partly loaded, it does not reveal
how vacant and loaded sites are distributed within
individual multi-enzymes. There is a pressing need to
develop MS protocols for analyzing larger proteins. It
may then be possible to study loading patterns in
intact dimeric multi-enzymes, or at least in fragments
that retain the homodimeric bonding that exists in the
intact systems.

Concluding remarks
Limited proteolysis followed by LC ⁄ ion-trap MS is a
powerful and convenient technique for establishing features of PKS catalysis that are not readily accessible
by other means. The discovery that the first condensation reaction on the DEBS becomes an additional
bottleneck with an unnatural starter acid provides a
rational basis for efforts to improve productivity.
Thus, alteration of the AT domain of the loading
module would be unlikely to remedy the limitation,
whereas replacement of the KS by one normally operating with longer acyl chains might do so. Turnover
on the DKS with its normal starter acyl unit is clearly
regulated by the rate of release of the diketide product
by the TE domain. Studies of the extent of loading of
multienzymes with more than one module are needed
to establish if product release is indeed a general basis
of regulation in PKS operations, especially in more
fully evolved systems, such as the DEBS. The proposal
that there are two forms of control resulting from high
levels of occupancy of active sites by intermediates,

congestion control and retardation control, is based on
the direct evidence that the KS domain is fully occupied under conditions of synthesis. The evidence for
the proposed quaternary effects, suppression of condensation and tandemization, is more circumstantial
but deserves further study.
MS also has the potential to play a major supporting role in structural studies of modular PKS multienzymes. It provides a method of quality control in
preparing samples for structural studies by NMR, elec7066

tron microscopy, or X-ray crystallography. This check
on structure will be particularly desirable in the preparation of derivatized forms of multienzymes that have
natural or unnatural ligands attached to the active sites
of selected domains.

Experimental procedures
Purification of the DEBS-derived DKS
The expression (in Escherichia coli) and purification of the
engineered DKS (comprising the first extension module of
DEBS1, covalently linked to the C-terminal TE domain
from DEBS 3) have been previously described [9,37]. In
this construct the extension module ACP (referred to here
as ACP1) is a hybrid of ACP1 and ACP6, in order to
preserve the native linker between the TE domain and the
ACP.

Limited proteolysis and LC ⁄ MS analysis
Limited proteolysis was performed at a protein ⁄ trypsin
ratio of 80:1 (w ⁄ w) at 30 °C for 5 min. After digestion,
the mixture was immediately injected onto a pre-equili˚
brated C4 column (4.6 · 250 mm, 300A; Vydac, Hesperia,
CA, USA) and proteins were eluted with a linear gradient of 35–55% acetonitrile (containing 0.1% trifluoroacetic acid) over 40 min. The analysis was performed using
online LC ⁄ MS on an ion-trap instrument (LCQ Classic;

ThermoFinnigan, San Jose, CA, USA). xcalibur 1.0
(ThermoFinnigan) software was used to operate the system, and bioworks 1.0 software (ThermoFinnigan) was
used for mass deconvolution. The detailed conditions for
limited proteolysis and LC ⁄ MS analysis have been
described previously [37].

Substrate specificity for the chain-extender unit
A 6 mm concentration of malonyl-CoA or (RS)-methylmalonyl-CoA was incubated with 6 lm DKS in a total
volume of 30 lL, containing 400 mm potassium phosphate (pH 7.4), 1 mm EDTA, 1 mm dithiothreitol and
20% glycerol. The reactions were carried out at 30 °C
for 10 min. After the incubation, samples were immediately subjected to tryptic digestion and analysed using
LC ⁄ MS.

Purification of commercial methylmalonyl-CoA
Commercial methylmalonyl-CoA was dissolved in distilled
deionized (MQ) water (Millipore, Billerica, MA, USA), and
loaded onto a reverse-phase C18 column (Prodigy C18,
4.6 · 250 mm, 5 l; Phenomenex, Torrance, CA, USA).
Methylmalonyl-CoA and propionyl-CoA were separated by

FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS


H. Hong et al.

Changing patterns of covalent active-site occupancy

a linear gradient of 5–20% acetonitrile, containing 0.1%
trifluoroacetic acid, over 20 min. Methylmalonyl-CoA was
eluted about 2 min before the propionyl-CoA. Fractions

containing methylmalonyl-CoA were pooled, lyophilized
and kept at )20 °C. Purified methylmalonyl-CoA was redissolved in MQ water before use, and kept on ice.

adjusted to pH 6–7 with formic acid and analyzed using
LC ⁄ MS. The analysis was performed using the protocol
described above except that after 20 min, the mass
spectrometer was set up in a single full-scan mode, with
scan range from m ⁄ z 600 to 2000, to monitor the ACPTE.

Observation of intermediates on the DKS
multienzyme

Acknowledgements

DKS (5 lm) was incubated with 2 mm acyl-CoA, 2 mm
methylmalonyl-CoA, and 6 mm NADPH in a total volume
of 50 lL, containing 400 mm potassium phosphate (pH
7.4), 1 mm EDTA, 1 mm dithiothreitol and 20% glycerol.
After reaction at 30 °C for 10 min, the DKS was subjected
to tryptic digestion and LC ⁄ MS analysis. Propionyl-CoA,
n-butyryl-CoA and n-valeryl-CoA were used individually to
supply starter units. When n-butyryl-CoA and n-valerylCoA were used, purified methylmalonyl-CoA was applied
to avoid minor contamination of propionyl-CoA in the
commercial methylmalonyl-CoA.

Off-loading and analysis of the b-OH diketide
from the diketide ACP-TE intermediate
The conditions for off-loading and analysis of the b-OH
diketide were optimized on a model system as follows. A
sample (2 nmol) of b-OH diketide-SNAC (prepared as

described previously [48]) was dissolved in 400 mm potassium phosphate buffer (pH 7.4). Then, 2 lL of hydrazine
was added to a total volume of 25 lL. After allowing the
reaction to proceed at room temperature for 1 h, the sample was adjusted to pH 6–7 with formic acid and the mixture was analyzed using LC ⁄ MS. The analysis was
performed on a C18 column (Prodigy C18, 2.0 · 250 mm,
5 l; Phenomenex) with a gradient of 2–50% acetonitrile
containing 0.1% trifluoroacetic acid, over 20 min. The
LCQ mass spectrometer was set up in two scan modes: full
scan mode scanning from m ⁄ z 50 to 200; and MS ⁄ MS
mode with m ⁄ z 147.1 as the precursor ion and collision
energy at 20.5%. Fractions containing the hydrazide reaction product were also collected, lyophilized and analyzed
on a Q-TOF (Micromass, Manchester, UK) high-resolution
mass spectrometer.
Following the assay of overall diketide formation (with
propionyl-CoA providing the starter unit), limited proteolysis and HPLC separation were performed as described
above for the model system. Fractions containing the
diketide ACP-TE were collected, combined and lyophilized. A total of 550 lg of DKS was used for generating
the acyl ACP-TE. The lyophilized protein was redissolved
in 400 mm potassium phosphate buffer (pH 7.4) and
5 lL of neat hydrazine was added to give a total volume
of 50 lL. The reaction was allowed to proceed at room
temperature for 1 h. The reaction mixture was then

We are grateful to Drs K.J. Weissman and A.M. Hill
for their helpful comments and suggestions. We also
gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council
(BBSRC) (UK) via a project grant to P.F.L. and the
late Dr J.B. Spencer (8 ⁄ B18119).

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