The changing patterns of covalent active site occupancy
during catalysis on a modular polyketide synthase
multienzyme revealed by ion-trap mass spectrometry
Hui Hong
1,2
, Peter F. Leadlay
2
and James Staunton
1
1 Department of Chemistry, University of Cambridge, UK
2 Department of Biochemistry, University of Cambridge, UK
Introduction
Polyketides are a large and diverse group of secondary
metabolites that are produced by a common biosyn-
thetic 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
Keywords
enzyme-bound intermediate; erythromycin;
limited proteolysis; liquid chromatography-
mass 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,

evidence for simultaneous loading of both acylcarrier proteins and for the
coordination of timing between the two active centres for chain extension.
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 ].
FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7057
the general structural ketene unit, RCH=C=O, until
the linear chain of carbons reaches the desired length.
Subsequent diverse biosynthetic transformations gener-
ate an enormously varied set of structures [1,2].
The catalytic enzymes responsible for the chain-exten-
sion processes show a remarkable degree of structural
and mechanistic homology across the wide range of bio-
synthetic organisms. There are large differences, how-
ever, 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-exten-
sion 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

OH
Me
O
OH
O
OH
OH
Me
O
O
OH
OH
Me
O
OH
OH
Me
O
OH
OH
Me
O
OH
Me
O
Me
O
S
S
S

ACP
ACP
DH
ER
Intermediates
DEBS1 DEBS2 DEBS3
6-Deoxyerythronolide B
(6-DEB)
Cyclise on
TE domain
Load Module 1 Module 2 Module 3 Module 4
Module 5 Module 6
SH
KS
KR
ATAT
ACP
OH
TE
ACP
OH
OH
O
Me
S
O
OH
Me
S
Hydrolyse

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 enzyme-
catalyzed steps [12], or even whole modules, are
‘skipped’ [13,14], while in other cases extension mod-
ules 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 prod-
uct [18–21]. Recent structural studies on the intact ani-
mal 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 multienzyme-
bound intermediates and to establish the pattern and
level of covalent attachment of substrates and interme-
diates to individual active sites, during catalysis in vitro
on a modular PKS. For dissociated (Type II) FAS and

the method to establish the pattern of covalent interme-
diates 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 methyl-
malonyl-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, cor-
responding to the form loaded with methylmalonate
(see Fig. 3). In this case, a ratio of approximately 55%

SH
SH
SH
KS
KR
ATAT
ACP
OH
TE
ACP
OH
OH
SH
OH
TE
ACP
OH
TEKRAT
OH
SH
KS
SH
AT
ACP
OH
S
SH
KS
KR
ATAT

ATAT
ACP
OH
TE
ACP
OH
OH
TE
ACP
OH
TEKR
AT
SH
KS
SH
AT
ACP
OH
Proteolysis
Proteolysis
Proteolysis
O
O
CO
2
H
S
O
CO
2

strate 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 estab-
lish 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
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 inter-
mediate from this domain is faster than its acylation
by diketide transfer from ACP1. Therefore any cova-
lently 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

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.
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. Methylmalonyl-CoA 45 55 N ⁄ AN⁄ A
2. Malonyl-CoA 0 0 N ⁄ AN⁄ A
3. Propionyl-CoA; methylmalonyl-CoA; NADPH 42 0 0 58
4. Butyryl-CoA; methylmalonyl-CoA; NADPH 51 0 0
a
49
a
5. Valeryl–CoA; methylmalonyl-CoA; NADPH 37 35 0
a
28
a
6. Propionyl-CoA; methylmalonyl-CoA; no NADPH 45 55 0 0
a
Absence of keto-diketide assumed by analogy with experiment 3.
H. Hong et al. Changing patterns of covalent active-site occupancy
FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7061
First, suitable conditions for the reaction were estab-
lished 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]

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. Incuba-
tion of DKS with valeryl-CoA likewise led to the com-
plete 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.
ACP
KS
KR
TE
ATAT
ACP
OH
SHSHSH
OH
OH
R
O
O
S
CO
2
H

39 611
Diketide
39 644
39 300 39 500 39 700 39 900
Mass (Da)
28 000 32 000 36 000 40 000
Mass (Da)
A
B
C
D
Fig. 5. MS results from experiments 3, 4 and 5. (A) Control experi-
ment without added precursors. (B) Incubation with propionate as
the starter in experiment 3. (C) Incubation with butyrate as the star-
ter in experiment 4. (D) Incubation with valerate as the starter in
experiment 5, showing the expanded version of the ACP-TE region.
Changing patterns of covalent active-site occupancy H. Hong et al.
7062 FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS
Finally, an experiment was carried out in which the
natural substrates propionyl-CoA and methylmalonyl-
CoA were supplied, but in which NADPH was not
supplied. The aim was to see if the keto-ester interme-
diate accumulated, and, if so, which stereoisomer dom-
inated 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 chain-
extension ACP (ACP1) in this experiment. There was a

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 trans-
fer of the diketide intermediate from the chain-exten-
sion 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 dike-
tide fell significantly, consistent with a slowing of the
rate of the condensation step relative to the offload-
ing step (we assumed that all the diketide intermedi-
ate was in the hydroxy form in experiments 4 and
5). In the valerate experiment there was a more dra-
matic change. The proportion of the diketide inter-
mediate fell even further (below 50%) and a
detectable amount of ACP1 was found to be loaded
with methylmalonate. It would appear that the con-

2
NHN
NH
2
NH
2
NH
2
NH
2
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.
H. Hong et al. Changing patterns of covalent active-site occupancy
FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7063
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 subse-
quently 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

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 recog-
nition 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].
Evidence for coordination of condensation and
ketoreduction
In previous work [44] the operation of a single exten-
sion module from DEBS was studied in vitro by mix-
ing 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 there-
fore 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

and the two ACP domains are able to approach
Changing patterns of covalent active-site occupancy H. Hong et al.
7064 FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 hori-
zontally 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 bio-
synthetic intermediates from the surrounding aqueous
medium. If the collar can expand and contract to con-
trol the lateral passage of the ACP domains, there
exists a possible mechanism by which the presence of
NADPH might enable the condensation step by bind-
ing 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

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 qua-
ternary structure, each ACP domain can access the appropriate
KS domain for the condensation step (and other domains in suc-
cession through the chain extension cycle) by moving backwards
and forwards (dashed green arrows) along the axis within the cen-
tral core of the structure. Because of the restricting effect of the
short ACP to the TE linker, and the anticipated need for co-ordi-
nated movements of the two looped-out domains, it is likely that
the pair of ACP domains move backwards and forwards in tan-
dem, rather than independently. As a result, the successive reac-
tions 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 KS-

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 fea-
tures of PKS catalysis that are not readily accessible
by other means. The discovery that the first condensa-
tion 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 oper-
ating 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

brated C4 column (4.6 · 250 mm, 300A
˚
; Vydac, Hesperia,
CA, USA) and proteins were eluted with a linear gradi-
ent of 35–55% acetonitrile (containing 0.1% trifluoroace-
tic 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 sys-
tem, 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
A6mm concentration of malonyl-CoA or (RS)-methyl-
malonyl-CoA was incubated with 6 lm DKS in a total
volume of 30 lL, containing 400 mm potassium phos-
phate (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 immedi-
ately 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
Changing patterns of covalent active-site occupancy H. Hong et al.
7066 FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS

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 reac-
tion 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 prote-
olysis and HPLC separation were performed as described
above for the model system. Fractions containing the
diketide ACP-TE were collected, combined and lyophi-
lized. 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
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 ACP-
TE.
Acknowledgements
We are grateful to Drs K.J. Weissman and A.M. Hill
for their helpful comments and suggestions. We also

8 Khosla C, Tang Y, Chen AY, Schnarr NA & Cane DE
(2007) Structure and mechanism of the 6-deoxyerythr-
onolide B synthase. Ann Rev Biochem 76, 195–221.
9 Østergaard LH, Kellenberger L, Corte
´
s J, Roddis MP,
Deacon M, Staunton J & Leadlay PF (2002) Stereo-
chemistry of catalysis by the ketoreductase activity in
the first extension module of the erythromycin polyke-
tide synthase. Biochemistry 41, 2719–2726.
10 Weissman KJ, Timoney M, Bycroft M, Grice P, Hane-
feld U, Staunton J & Leadlay PF (1997) The molecular
basis of Celmer’s rules: the stereochemistry of the con-
densation step in chain extension on the erythromycin
polyketide synthase. Biochemistry 36, 13849–13855.
H. Hong et al. Changing patterns of covalent active-site occupancy
FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7067
11 Walsh C & Cane DE (1999) The parallel and conver-
gent universes of polyketide synthases and nonriboso-
mal peptide synthetases. Chem Biol 6, R319–R325.
12 Kellenberger L, Galloway IS, Sauter G, Bo
¨
hm G,
Hanefeld U, Corte
´
s J, Staunton J & Leadlay PF (2008)
A polylinker approach to reductive loop swaps in mod-
ular polyketide synthases. ChemBioChem 9, 2740–2749.
13 Rowe CJ, Bo
¨

ller R (2002) The biosynthesis
of the aromatic myxobacterial electron transport inhibi-
tor stigmatellin is directed by a novel type of modular
polyketide synthase. J Biol Chem 277, 13082–13090.
19 He J & Hertweck C (2003) Iteration as programmed
event during polyketide assembly: molecular analysis of
the aureothin biosynthetic gene cluster. Chem Biol 10,
1225–1232.
20 Olano C, Wilkinson B, Sanchez C, Moss SJ, Sheridan
RM, Math V, Weston AJ, Brana AF, Martin CJ, Oliy-
nyk M et al. (2004) Biosynthesis of the angiogenesis
inhibitor borrelidin by Streptomyces parvulus Tu
¨
4055:
cluster analysis and assignment of functions. Chem Biol
11, 87–97.
21 Tatsuno S, Arakawa K & Kinashi H (2007) Analysis of
modular-iterative mixed biosynthesis of lankacidin by
heterologous expression and gene fusion. J Antibiot 60,
700–708.
22 Maier T, Leibundgut M & Ban N (2008) The crystal
structure of a mammalian fatty acid synthase. Science
321, 1315–1322.
23 Brignole EJ, Smith S & Asturias FJ (2009) Conforma-
tional flexibility of metazoan fatty acid synthase enables
catalysis. Nat Struct Mol Biol 16, 190–197.
24 Tang Y, Kim CY, Mathews II, Cane DE & Khosla C
(2006) The 2.7-Angstrom crystal structure of a 194-kDa
homodimeric fragment of the 6-deoxyerythronolide B
synthase. Proc Natl Acad Sci U S A 103, 11124–11129.

Chem 9, 150–156.
32 Mazur MT, Walsh CT & Kelleher NL (2003) Site-
specific observation of acyl intermediate processing in
thiotemplate biosynthesis by Fourier Transform mass
spectrometry: The polyketide module of yersiniabactin
synthetase. Biochemistry 42, 13393–13400.
33 Hicks LM, O’Connor SE, Mazur MT, Walsh CT &
Kelleher NL (2004) Mass spectrometric interrogation
of thioester-bound intermediates in the initial stages
of epothilone biosynthesis. Chem Biol 11, 327–
335.
34 Dorrestein PC & Kelleher NL (2006) Dissecting non-
ribosomal and polyketide biosynthetic machineries using
electrospray ionization Fourier-Transform mass spec-
trometry. Nat Prod Rep 23, 893–918.
35 Bumpus SB & Kelleher NL (2008) Accessing natural
product biosynthetic processes by mass spectrometry.
Curr Opin Chem Biol 12, 475–482.
36 Aparicio JF, Caffrey P, Marsden AF, Staunton J &
Leadlay PF (1994) Limited proteolysis and active-site
studies of the first multienzyme component of the eryth-
Changing patterns of covalent active-site occupancy H. Hong et al.
7068 FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS
romycin-producing polyketide synthase. J Biol Chem
269, 8524–8528.
37 Hong H, Appleyard AN, Siskos AP, Garcia-Bernardo
J, Staunton J & Leadlay PF (2005) Chain initiation on
type I modular polyketide synthases revealed by limited
proteolysis and ion-trap mass spectrometry. FEBS J
272, 2373–2387.

ketide synthases. Chem Biol 15, 1231–1240.
44 Castonguay R, He W, Chen AY, Khosla C & Cane DE
(2007) Stereospecificity of ketoreductase domains of the
6-deoxyerythronolide B synthase. J Am Chem Soc 129,
13758–13769.
45 Bo
¨
hm I, Holzbaur IE, Hanefeld U, Corte
´
s J, Staunton
J & Leadlay PF (1998) Engineering of a minimal modu-
lar polyketide synthase, and targeted alteration of the
stereospecificity of polyketide chain extension. Chem
Biol 5, 407–412.
46 Staunton J, Caffrey P, Aparicio JF, Roberts GA,
Bethell SS & Leadlay PF (1996) Evidence for a double-
helical structure for modular polyketide synthases.
Nat Struct Biol 3, 188–192.
47 Kao CM, Pieper R, Cane DE & Khosla C (1996)
Evidence for two catalytically independent clusters of
active sites in a functional modular polyketide synthase.
Biochemistry 35, 12363–12368.
48 Harris RC, Cutter AL, Weissman KJ, Hanefeld U,
Timoney MC & Staunton J (1998) Enantiospecific
synthesis of analogues of the diketide intermediate of
the erythromycin polyketide synthase. J Chem Res (S),
283.
H. Hong et al. Changing patterns of covalent active-site occupancy
FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7069