Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap...

Chain initiation on type I modular polyketide synthasesrevealed by limited proteolysis and ion-trap massspectrometryHui Hong1, Antony N. Appleyard2, Alexandros P. Siskos2, Jose Garcia-Bernardo2, James Staunton1

and Peter F. Leadlay2

1 Department of Chemistry, University of Cambridge, UK

2 Department of Biochemistry, University of Cambridge, UK

Polyketides are a structurally diverse group of natural

products, which exhibit a broad range of biological

effects including antibiotic, antifungal, immunosup-

pressive, and anticancer activities [1]. They are synthes-

ized on polyketide synthases (PKSs), which convert

intracellular acyl-CoA precursors into complex poly-

ketide backbones via a stepwise chain building mech-

anism employing different combinations of a standard

set of biochemical reactions. There are three canonical

types of PKS, based on their structure and mecha-

nisms of operation: type I (iterative or modular),

type II and type III [2]. The best-studied modular

type I PKS is the 6-deoxyerythronolide B synthase

(EC 2.3.1.94) (DEBS) from Saccharopolyspora erythr-

aea, which produces the polyketide backbone of the

antibiotic erythromycin (Fig. 1A). DEBS consists of

three large bimodular polypeptides (DEBS1, DEBS2,

and DEBS3) (each >300 kDa) which together catalyze

the stepwise condensation of a propionyl-CoA-derived

primer unit with six methylmalonyl-CoA-derived exten-

der units to yield 6-deoxyerythronolide B (6dEB) [1].

The hallmark of a modular type I PKS is that there is a

separate domain for every step of the assembly of the

polyketide chain, and they are disposed along the PKS

Keywords

erythromycin; limited proteolysis; liquid

chromatography-mass spectrometry;

multienzyme; polyketide synthase

Correspondence

J. Staunton, Department of Chemistry,

University of Cambridge, Lensfield Road,

Cambridge CB2 1EW, UK

Fax: +44 1223 762018

Tel: +44 1223 766041

E-mail: [email protected]

(Received 10 November 2004, revised 28

January 2005, accepted 15 February 2005)

doi:10.1111/j.1742-4658.2005.04615.x

Limited proteolysis in combination with liquid chromatography-ion trap

mass spectrometry (LC-MS) was used to analyze engineered or natural

proteins derived from a type I modular polyketide synthase (PKS), the

6-deoxyerythronolide B synthase (DEBS), and comprising either the first

two extension modules linked to the chain-terminating thioesterase (TE)

(DEBS1-TE); or the last two extension modules (DEBS3) or the first exten-

sion module linked to TE (diketide synthase, DKS). Functional domains

were released by controlled proteolysis, and the exact boundaries of

released domains were obtained through mass spectrometry and N-terminal

sequencing analysis. The acyltransferase-acyl carrier protein required for

chain initiation (ATL-ACPL), was released as a didomain from both

DEBS1-TE and DKS, as well as the off-loading TE as a didomain with the

adjacent ACP. Mass spectrometry was used successfully to monitor in

detail both the release of individual domains, and the patterns of acylation

of both intact and digested DKS when either propionyl-CoA or n-butyryl-

CoA were used as initiation substrates. In particular, both loading domains

and the ketosynthase domain of the first extension module (KS1) were

directly observed to be simultaneously primed. The widely available and

simple MS methodology used here offers a convenient approach to the pro-

teolytic mapping of PKS multienzymes and to the direct monitoring of

enzyme-bound intermediates.

Abbreviations

ACP, acyl carrier protein; AT, acyl transferase; DEBS, 6-deoxyerythronolide B synthase; DKS, diketide synthase; KR, ketoreductase; KS,

ketosynthase; NPDS, 4-nitrophenyl disulfide; NRPS, nonribosomal peptide synthase; PKS, polyketide synthase; TE, thioesterase.

FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 2373

multienzyme polypeptides essentially in the order that

they are used.

Modular PKSs are clearly amenable to rational gen-

etic manipulation of the biosynthetic enzymes, as a

promising way of creating new bioactive compounds

[3,4]. However to achieve this efficiently we need a

better understanding of the molecular basis underlying

the operation of these assembly line enzymes. To facili-

tate the detailed mechanistic study of the erythromycin

biosynthesis, model systems with shortened length have

been created. DEBS1-TE is a bimodular PKS, created

by moving the thioesterase (TE) domain from the ter-

minus of DEBS3 to the end of DEBS1 to cause prema-

ture release of the chain at the triketide stage (Fig. 1B)

[5]. The unimodular PKS, called diketide synthase

(DKS) was created by moving the TE domain from

the terminus of DEBS3 to the end of module 1 of

DEBS1, to cause premature release of the chain at the

diketide stage (Fig. 1C) [6]. It should be noted that the

engineering of these model proteins was designed to

preserve the native linker between the TE domain and

the adjacent acyl carrier protein (ACP). The ACP

domains are therefore hybrid structures comprising the

N-terminal of ACP2 (DEBS1-TE) and ACP1 (DKS),

respectively, fused to the C-terminal portion of ACP6.

(The domain number is the module number in which

the domain resides. This designation applies through

out the paper.) For simplicity in the following account,

these hybrid ACPs are designated ACP2 and ACP1,

respectively. The engineered proteins, DEBS1-TE and

DKS, have been purified to homogeneity and have

produced the expected products in vitro [6,7], and

therefore can serve as convenient models for the full

DEBS system.

A

B

C

Fig. 1. Organization of DEBS multienzyme proteins. (A) Organization of DEBS from S. erythraea, which catalyses the biosynthesis of 6-deoxy-

erythronolide B. DEBS consists of three large bimodular polypeptides DEBS1, DEBS2, and DEBS3. DEBS3 contains module 5, module 6 and

the TE. (B) Recombinant bimodular protein DEBS1-TE was created by moving the TE domain from the terminus of DEBS3 to the end of

DEBS1 to cause premature release of the chain at the triketide stage. (C) Recombinant unimodular protein DKS was created by moving the

TE domain from the terminus of DEBS3 to the end of module 1 of DEBS1 to cause premature release of the chain at the diketide stage. AT,

acyl transferase; ACP, acyl carrier protein; KS, ketosynthase; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; TE, thioesterase.

Limited proteolysis and MS of modular PKSs H. Hong et al.

2374 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS

Multifunctional proteins are generally organized into

structural domains in which contiguous regions of the

polypeptide are folded into separate globular units,

each having specific functions. The domains are con-

nected by short, flexible, surface-exposed linker regions

which are especially susceptible to proteolysis [8]. Lim-

ited proteolysis has proved to be very useful in the

study of the structure, assembly and mechanism of

multifunctional proteins [9–12]. We have previously

made extensive use of limited proteolysis in the study

of DEBS proteins [13,14], including the use of radio-

labelled substrates to probe the effects of proteolysis

on enzymatic activity. Unfortunately, radiolabelling

methods can give misleading results [15], and in addition

this technology does not provide detailed information

on the exact chemical form of the labelled complex.

Over the last 10 years, mass spectrometry has played

an increasingly important role in the study of biologi-

cal systems, because of its high sensitivity, accuracy

and speed. Recently, Fourier transform mass spectro-

metry (FTMS) has been used successfully in the

observation of different acyl-ACP intermediates in

yersiniabactin [16] and also in epothilone biosynthesis

mixed PKS-nonribosomal peptide synthetases (NRPSs)

[17]. There are, however, significant limitations on the

size of protein fragments suitable for FTMS analysis

[16], and so to obtain specific information on domains

other than the ACP (� 11 kDa), they need to be diges-

ted extensively into smaller peptides.

Here, we show that entire functional domains from

modular type I PKSs can be released and detected

by controlled limited proteolysis in combination with

on-line liquid chromatography-mass spectrometry

(LC-MS) analysis. Domain identities as well as the exact

domain boundaries are obtained. The domains released

by proteolysis retain their intrinsic activity, and the

acylation details of the DEBS loading module as well as

KS1 domain have been observed directly using relatively

simple and affordable ion trap mass spectrometry. The

reduced resolving power is compensated for by the

increase of detectable size (over 79 kDa in this study) in

the proteins. We have used these protocols to make

direct observations of bound starter units on the DEBS

proteins. The methodology, which is sensitive, specific

and convenient, provides an additional and powerful

tool in the study of modular PKSs and NRPSs.

Results

Limited proteolysis of DEBS1-TE

DEBS1-TE was digested with trypsin at several different

weight ratios at 30 �C, as described under Experimental

procedures, and for various lengths of time. The pro-

gress of the reaction was monitored using LC-MS analy-

sis. Optimal digestion was achieved at a protein ⁄ trypsinratio in the range from 50 : 1 to 100 : 1 (w ⁄w) at 30 �Cfor 5 min. A typical LC trace of tryptic digestion at a

protein ⁄ trypsin ratio of 75 : 1 is shown in Fig. 2A. The

masses corresponding to each peak are shown in

Table 1. In some cases, one or more fragments of differ-

ent mass were obtained for a particular region of the

protein due to the existence of more than one available

cleavage site in the adjacent linker region. The existence

of the multiple cleavage sites is useful in that they pro-

vide confirmation of the domain identity assignments.

To locate the precise position and the identity of the

released polypeptides, the observed masses were used to

search for the tryptic fragments from the known

DEBS1-TE amino acid sequence using the program

paws. The identity of individual peptide fragments was

further confirmed by automated N-terminal analysis.

With the exception of a 150 kDa fragment, which was

too large for its mass to be determined reliably, all the

Fig. 2. LC separation of fragments after limited proteolysis of

DEBS1-TE, DEBS3 and DKS. Fragments were detected by their

absorbance at 214 nm. Peaks relating to individual fragments are

labelled with their retention time and deduced identity. (A) Tryptic

digestion of DEBS1-TE at a protein–trypsin ratio of 75 : 1 (w ⁄w) at

30 �C for 5 min. (B) Tryptic digestion of DEBS3 at a protein–trypsin

ratio of 75 : 1 (w ⁄w) at 30 �C for 5 min. (C) Tryptic digestion of

DKS at a protein–trypsin ratio of 800 : 1 (w ⁄w) at 30 �C for 60 min.

Digestion of DKS at a protein–trypsin ratio of 80 : 1 (w ⁄w) at 30 �Cfor 5 min gave the same digestion pattern. Proteolytic fragments

were separated on a C4 reversed-phase column (Vydac, Protein

C4, 4.6 · 250 mm, 300 A) and eluted with a linear gradient from

35% to 55% acetonitrile (0.1% trifluoroacetic acid) ⁄water (0.1% tri-

fluoroacetic acid) over 40 min at a flow rate of 0.7 mLÆmin)1. LM,

loading module fragment comprising the didomain ATL-ACPL;

ACP1-M2, tetradomain fragment containing domains ACP1-KS2-

AT2-KR2; KR5-ACP5-M6, multidomain fragment containing KR5,

ACP5 and all or part of module 6.

H. Hong et al. Limited proteolysis and MS of modular PKSs


other fragments were detected with a mass accuracy of

0.01%, and therefore could be matched uniquely to the

amino acid sequence. Thus, both the N-terminal and the

exact C-terminal of the fragments as well as their identi-

ties were assigned (Table 1). Despite the uncertainty in

mass of the 150 kDa fragment, it was possible to con-

firm by N-terminal sequencing that this fragment starts

with ACP1, and based on the size of the observed mass

it probably comprises all of module 2 bar the C-terminal

ACP2 domain. However, ACP2 was observed separately

as part of the ACP2-TE didomain. Good LC separation

was achieved with the exception of the ACP2-TE and

TE fragments, which coelute. The digestion pattern of

DEBS1-TE generated by trypsin is in good agreement

with previous results from the tryptic digestion of

DEBS1 [13]. The loading module was released as a sta-

ble didomain ATL-ACPL. KR1 and TE were also both

released as stable single domains. Most of module 2

remained intact and did not release isolated domains

even when the protein was treated with up to 2 m urea

with the aim of partially unfolding the protein. To check

whether other proteinases could digest module 2, ela-

stase was also used to analyze DEBS1-TE. The resulting

digestion pattern from elastase was very similar to that

obtained following tryptic digestion (data not shown),

and again module 2 remained largely intact. Import-

antly, however, KS1 and AT1 were found to be released

as separate individual domains, which was in contrast to

the previous proteolysis results on DEBS1 and DKS,

where the KS1 and AT1 were always observed together,

either as a KS1-AT1 didomain or as part of larger pro-

teolytic fragments [6,13]. The ACP2 domain once

released seems to be susceptible to further proteolysis,

as it was never observed independently under the dig-

estion conditions employed, only as the ACP2-TE

didomain. Under harsher digestion conditions, even

ACP2-TE was degraded further leaving only the TE

domain intact. These observations suggest that the

ACP2 domain is stabilized by the presence of the TE

domain, as observed for PCP or ACP domains in other

NRPS and PKS proteins [12]. In contrast to the ACP2-

TE didomain, the loading didomain ATL-ACPL seemed

to be more resistant to proteolysis, and individual

domains were not observed, suggesting a strong inter-

action between the two domains. The correct post-trans-

lational modification with a 4¢-phosphopantetheinylprosthetic group of both the loading and extender ACPs

was confirmed by the fact that the observed mass of

ATL-ACPL and ACP2-TE could only be matched from

the DEBS1-TE amino acid sequence if the phospho-

pantetheinyl moiety is presumed to be present on both

ACPs (the calculated mass increase for addition of a

phosphopantetheinyl group is 339 Da).

Limited proteolysis of DEBS3

Purified DEBS3 was subjected to tryptic digestion as

described in Experimental procedures. Digestions were

carried out at two different protein ⁄ trypsin ratios,

250 : 1 (w ⁄w) and 75 : 1 (w ⁄w), but the resulting diges-

Table 1. Fragments identified after limited proteolysis of DEBS1-TE.

Fragment identity Corresponding sequence N-Terminal sequencea Observed mass (Da)b Expected mass (Da)

TE E3468-S3738 EASSALRDGY 28951 ± 1 28952

L3452-S3738 LAD**G 30610 ± 1 30613

R3451-S3738 RLA 30766 ± 1 30769

ACP2-TE A3363-S3738 AGEPETESLR 40592 ± 2 40255(apo)

40594 (holo)

KS1 T550-R1137 TNEAAPGEP 61196 ± 2 61200

A548-R1137 ARTNEAAPG 61424 ± 3 61428

AT1 E1138-R1418 EQDAALSTER 29770 ± 1 29770

E1138-R1429 31124 ± 1 31126

E1138-R1441 32588 ± 1 32587

ATL-ACPL T11-R544 TAQPGRIVRP 56003 ± 2 55667(apo)

56006 (holo)

T11-R547 56391 ± 2 56053(apo)

56392 (holo)

ACP1-KS2-AT2-KR2 V1925-R3362 VGALAS*PA 150114 ± 43 149829 (apo)

150168 (holo)

KR1 S1443-R1914 STEVDEVSAL 49642 ± 2 49647

R1442-R1914 RSTEVDEVS 49799 ± 3 49803

a All cleavages were at C-terminal of R residues (K is absent from the linker regions). b The error bars reported are based on at least three

independent experiments. *Signifies an unidentified residue.



tion pattern was the same in both cases. The LC trace

obtained for the digestion mixture at a protein ⁄ trypsinratio of 75 : 1 (w ⁄w) is shown in Fig. 2B. Only AT5

and ACP5 from module 5 and the TE domain were

observed as stable single domains. Their identities were

confirmed by both mass matching and N-terminal

sequencing analysis (Table 2). No single domain from

module 6 was observed. However, a large fragment

(greater than 100 kDa) was detected with a retention

time of 39.62 min. The identification of this fragment

was complicated by a neighbouring peak (retention time

of 38.82 min, observed mass of 57 195 Da), which

proved to arise from the E. coli chaperone protein

GroEL as judged by N-terminal sequencing and mass

spectrometric analysis. The 39.62-min polypeptide was

identified as beginning with KR5 by N-terminal sequen-

cing. Due to its large size and the relatively weak mass

spectrometric intensity, the exact C-terminus for this

fragment could not be identified. However, the approxi-

mate mass and the N-terminal sequencing results sug-

gested that this proteolytic fragment comprises KR5,

ACP5, and most or all of module 6. The didomain

ACP6-TE was not observed, but the TE domain itself

was obtained, with the same cleavage sites as observed

for DEBS1-TE. The release of ACP5 is significant in

that it is the only single ACP domain released in detect-

able quantities from the DEBS proteins. The observed

mass of ACP5 confirmed that it was in the apo form

without the phosphopantetheinyl prosthetic group

attached, as expected for the DEBS3 protein purified

from E. coli, which does not house a phosphopanthei-

nyltransferase active against DEBS [18,19]. In contrast,

DEBS1-TE and DKS, which were expressed in S. erythr-

aea, are expected to be in their holo forms.

Limited proteolysis of DKS

Purified DKS was subjected to limited tryptic pro-

teolysis under various conditions as described in

Experimental procedures. Domain and multidomain

fragments were reproducibly obtained when digestion

was carried out at a DKS ⁄ trypsin ratio of 800 : 1

(w ⁄w) at 30 �C for 1 h. In order to release the domains

rapidly for analysis following the acylation of DKS

(see later), a shorter digestion protocol was also inves-

tigated. We found that a 5-min digestion using a

DKS ⁄ trypsin ratio of 80 : 1 (w ⁄w) at 30 �C resulted in

the same digestion pattern as that from a 1-h digestion

at a DKS–trypsin ratio of 800 : 1 (w ⁄w). A typical LC

chromatogram of the proteolysed fragments from

DKS is shown in Fig. 2C. The masses corresponding

to each of the fractions are shown in Table 3. The pre-

cise location and identity of each digestion fragment

were assigned by mass mapping in combination with

N-terminal sequencing, and these data are also shown

in Table 3. The results were comparable to those of

DEBS1-TE in that all domains could be separated by

chromatography with the exception of the TE and

ACP1-TE fragments, which coeluted. Under the condi-

tions used, all the domain subunits from the DKS were

released either as individual domains or as a pair of

domains. The loading module was released as the sta-

ble didomain ATL-ACPL, and was resistant to further

digestion. KR1 and TE were released as stable indivi-

dual domains. Similarly, KS1 and AT1 were released

as individual domains (the deconvoluted mass spectra

for ATL-ACPL and KS1 are shown in Fig. 3A and

Fig. 4A, respectively). As for ACP2 in DEBS1-TE,

ACP1 was apparently too susceptible to further pro-

teolysis for it to be observed. The ACP1-TE didomain

could be observed under milder digestion conditions.

The complete post-translational modification of both

the loading and extender ACPs was also confirmed by

the observed masses.

Propionyl-CoA/n-butyryl-CoA incubation with

intact and digested DKS

The acyl-CoA substrates were incubated either with

intact protein or with the mixture of domain fragments

Table 2. Fragments identified after limited proteolysis of DEBS3. * Signifies an unidentified residue.


ACP5 Q1368-R1478 QSEEGPALAQ 12 006 ± 1 12 005(apo)

TE E2021-S2291 EASSALRDGY 28 950 ± 1 28 952

L2005-S2291 LADHIGQQ 30 611 ± 2 30 613

R2004-S2291 RL*DH 30 766 ± 1 30 769

AT5 T549-R894 TRRGVAMVF 36 676 ± 1 36 679

KR5-ACP5-M6 A907-? ARDEDDD*RY > 100 000

a All cleavages were at C-terminal of arginine residues (lysine is absent from the linker regions). b The error bars reported are based on at

least three independent experiments.



released by limited proteolysis, to detect any differ-

ences in acylation behaviour. (Overall polyketide syn-

thase activity was not measured.) For example, if

certain domains only become acylated via transfer of

starter units from tethered adjacent domains, they

might fail to be labelled in the mixture of fragments.

Intact DKS

The ability to release and obtain the precise mass of

individual domains and domain pairs from the DKS

enables the study of the acylation specificity for each

individual AT and ACP, as well as KS1 domains of

this multidomain enzyme.

Propionyl-CoA, the native substrate for the DEBS

loading module, was incubated with the intact DKS at

30 �C for 10 min, followed by a 5-min tryptic digestion

to release domains for analysis (Fig. 5B). Analysis of

the mass of each peak revealed that propionyl units

were specifically loaded onto fragments ATL-ACPL

and KS1 but not onto AT1, KR1, ACP1 or TE

domains. This clearly confirms that propionyl-CoA is

not a substrate for the extender AT1 and ACP1

domain. More significantly, after incubation with pro-

pionyl-CoA, the LC trace for the loading module frag-

ment showed two peaks, designated LM1 and LM2,

with a mass increase of 55 and 111 Da, respectively,

which within the experimental error corresponds to

loading of one and two propionyl units, respectively

(theoretical mass increase of 56 and 112 Da, respect-

ively) (Fig. 3B,C). No unacylated ATL-ACPL was

observed. The observation of a mass increase of

111 Da directly confirms that both active sites in the

loading didomain may be simultaneously acylated.

KS1 was also fully acylated by the incubation with

propionyl-CoA, with a mass increase of 55 Da, and no

residual free KS1 was observed (Fig. 4B). Similar

results were obtained when intact DEBS1-TE was trea-

ted with propionyl-CoA prior to digestion (data not

shown). So, for the first time, a stoichiometric binding

of the substrate on the DEBS loading module as well

as on the KS1 has been directly observed.

When the alternative non-natural substrate n-butyryl-

CoA, which also progressed to full-length polyketide

[20], was incubated with the intact DKS, similar results

were obtained (Fig. 5C). Like propionyl-CoA, the buty-

ryl group was specifically loaded onto fragment ATL-

ACPL and KS1 but not onto AT1, KR1, ACP1 or TE.

The loading module fragment also showed two peaks,

LM1 and LM2 with mass increase of 67 and 137 Da

(theoretical mass increase of 70 and 140 Da, respect-

ively), which corresponds to single and double acylation

by the butyryl group, respectively (Fig. 3D,E). KS1 was

also fully acylated by the butyryl group with a mass

increase of 68 Da (Fig. 4C). No residual free ATL-

ACPL and KS1 were observed. The results not only

provide direct evidence that the DEBS loading module

possesses flexible substrate specificity, which is in agree-

ment with previous radiolabelling studies [21], but also

demonstrate that the mass accuracy in our experiments

is sufficient to distinguish between propionyl and buty-

ryl groups even for a protein over 60 kDa.

Table 3. Fragments identified after limited proteolysis of DKS. *Signifies an unidentified residue.


TE E2021-S2291 EASSALRDGY 28 950 ± 1 28 952

L2005-S2291 LADH*GQQ 30 610 ± 2 30 613

R2004-S2291 RLADHI*QQ 30 766 ± 1 30 769

ACP1-TE V1925-S2291 VGALTGLPR 39 507 ± 1 39 171(apo)

39 510 (holo)

KS1 T550-R1137 TNEAAPG 61 195 ± 2 61 200

A548-R1137 ARTNEA 61 422 ± 2 61 428

AT1 E1138-R1418 EQDAALSTER 29 768 ± 1 29 770

E1138-R1429 31 124 ± 1 31 126

E1138-R1441 32 585 ± 1 32 587

E1138-R1442 32 742 ± 1 32 744

ATL-ACPL T11-R544 TAQPGRIVRP 56 003 ± 2 55 667(apo)

56 006 (holo)

T11-R547 56 389 ± 3 56 053(apo)

56 392 (holo)

KR1 S1443-R1914 STEVDEVS 49 642 ± 2 49 647

R1442-R1914 RSTEVDEVS 49 798 ± 2 49 803

a All cleavages were at C-terminal of R residues (K is absent from the linker regions). b The error bars reported are based on at least three

independent experiments.



Digested DKS

To check whether the domains released from the DKS

retain their catalytic activities after proteolysis, propio-

nyl-CoA and n-butyryl-CoA were also individually

incubated with predigested DKS at 30 �C for various

lengths of time. The maximum level of acylation was

found after 10-min incubation (data not shown). Care-

ful comparison of the LC traces as well as the acyla-

tion details of each domain revealed no discernible

difference between the acylation patterns when either

propionyl-CoA or n-butyryl-CoA were used, before or

after proteolysis. The loading module was either singly

or doubly acylated by the propionyl- or n-butyryl-

CoA, and no unacylated loading module was observed.

KS1 was also fully acylated by either substrate, while

no acylation was observed on other domains. The

results suggest that domains maintain the same intrin-

sic catalytic activity whether in isolation or within the

quaternary structure of an intact DEBS module.

Fig. 3. LC separation of fragments from trypsin-digested DKS and detection of acyl-enzymes. Fragments were detected through their absorb-

ance at 214 nm. Fragments are shown from tryptic digestion of (A) DKS (control); (B) DKS, followed by incubation with propionyl-CoA; (C)

DKS, followed by incubation with n-butyryl-CoA; (D) DKS, followed by incubation with thiol-directed reagent NPDS; (E) DKS, pretreated with

NPDS, and after digestion incubated with propionyl-CoA. The identity of domains present in each peak is indicated, together with their inferred

acylation status. Separation conditions are the same as in Fig. 2. In D and E, the first peak contains TE only, and the ACP1-TE is present as a

disulfide bond-linked dimer indicated by the arrow. *LM, loading module comprising ATL-ACPL; LM1 and LM2, signify singly and doubly acylat-

ed loading module, respectively; LM(S-S), loading module containing an internal disulfide bond between the ATL and the phosphopantetheine

of ACPL; LM1(S-S), singly acylated loading module containing an internal disulfide bond between the ATL and the phosphapantetheine of

ACPL. �It is not known whether the single acyl group is attached exclusively to the active site of ATL or of ACPL, or both.



Probing the sites of acylation of loading

didomain with 4-nitrophenyl disulfide

Previous experiments with apo DEBS loading module

using radiolabelling showed that the extent of labelling

was about half that when holo protein was used, as

expected, as the loading module has two active sites,

and the phosphopantetheinyl prosthetic group is

required for attachment of the substrate to the ACP

domain [21]. We wished to use mass spectrometry as an

analytical tool directly to probe the involvement of

phosphopantetheine by using a thiol-modifying reagent

4-nitrophenyl disulfide (NPDS) which reacts with sul-

fhydryl groups at neutral pH. The trypsin-digested DKS

was treated with an excess of NPDS at 30 �C for 5 min,

followed by LC-MS analysis (Fig. 5D). Comparison of

the digested DKS before and after the treatment of

NPDS showed that after NPDS treatment, the first elut-

ed peak no longer contained the ACP1-TE didomain,

only the TE domain. However, an extra peak was eluted

between the TE and the KS1, and had a molecular mass

of 79013 Da. N-terminal sequencing analysis showed

that it corresponded to the ACP1-TE. Therefore, it most

likely corresponds to a disulfide bond-linked dimer of

ACP1-TE, which has an expected mass of 79018 Da.

Unexpectedly, the loading module seemed unaffected by

NPDS, since no mass increase was observed. In addition,

careful analysis of each eluted peak showed no evidence

A

B

Fig. 4. Effect of 4-nitrophenyl disulfide

treatment on the electrospray mass spec-

trum of the loading didomain ATL-ACPL.

(A) Mass spectrum of the loading didomain

ATL-ACPL resulting from tryptic digestion of

DKS; (B) mass spectrum of the loading

didomain ATL-ACPL resulting from tryptic

digestion of DKS, after subsequent treat-

ment with NPDS. The formation of an inter-

nal disulfide bond between the ATL and

ACPL, induced by NPDS treatment, results

in alteration of the m ⁄ z distribution to a

higher mass range (see text for details).



for a disulfide bond-linked dimer of ATL-ACPL. How-

ever, incubation of propionyl-CoA with digested DKS,

which had been pretreated with NPDS, resulted in the

formation of only singly acylated loading module with

a mass increase of 54 Da [Fig. 5E, peak labelled as

LM1(S-S)], with no doubly acylated form being

observed. This indicated that the thiol of the phospho-

pantetheine of the ACPL was blocked by the treatment

Fig. 5. Deconvoluted mass spectra of loading didomain ATL-ACPL released from DKS by limited proteolysis. (A) unliganded loading module;

(B) and (C), loading didomain, respectively, singly and doubly acylated after incubation with propionyl-CoA either before or after proteolysis;

(D) and (E), loading didomain, respectively, singly and doubly acylated after incubation with n-butyryl-CoA either before or after proteolysis.



of NPDS, and only the active site serine residue of ATL

was left available for acylation. When the mass spectra

of the NPDS-treated and untreated loading module

were compared, the m ⁄ z distribution pattern showed

significant differences (Fig. 6A,B). The m ⁄ z envelope of

peaks shifted to higher values after the NPDS treatment,

indicating that an intramolecular disulfide bond might

have formed within the loading didomain (the mass

accuracy for the 56 kDa protein would not allow us to

detect the 2 Da mass decrease due to the formation of

such an internal disulfide bond). The formation of the

intramolecular disulfide bond would make the protein

more compact, therefore leaving fewer chargeable sites

available for electrospray ionization, which resulted in

higher m ⁄ z-values in the spectrum. To confirm that an

intramolecular disulfide bond had formed within the

loading didomain, the reducing reagent dithiothreitol

was added in excess to the NPDS pretreated digestion

mixture, before the mixture was analyzed using LC-MS.

As expected, the m ⁄ z distribution of the loading module

shifted back to its original position, suggesting that the

internal disulfide bond was reduced by dithiothreitol

(data not shown). Once the excess dithiothreitol in the

sample was removed, double acylation of the loading

module was observed again with a mass increase of

109 Da (a theoretical mass increase 112 Da, data not

shown), upon addition of propionyl-CoA. Taken

together, these experiments provide evidence that the

thiol of the phosphopantetheinyl arm of ACPL is

involved in the priming of the substrate. When propio-

nyl-CoA was incubated with digested DKS, which had

been pretreated with NPDS, KS1 was still fully acylated,

confirming that NPDS does not affect the active site cys-

teine of KS1. This activity can be attributed to KS1 self-

acylation. However, around 20% of the loading

didomain was found to be unacylated [Fig. 5E, peak

labelled as LM(S-S)], which was in contrast to the full

acylation without NPDS treatment. The 20% unacy-

lated product is probably due to the hydrolysis of an ini-

tially formed mono-acyl-intermediate. It was reported

previously that when the apo DEBS loading didomain

was incubated with [14C]propionyl-CoA, following an

initial burst of radioactivity, a gradual decrease was

observed. The decrease of radioactivity was attributed

by the authors to the progressive hydrolysis of the

labelled substrates from the ATL [21].

Discussion

DEBS1-TE, DEBS3 and DKS were subjected to limited

tryptic digestion, and the digestion conditions were

optimized for each protein so that domains rather than

unstructured peptides were released from modules. This

Fig. 6. Deconvoluted mass spectra of KS1 released from DKS by

limited proteolysis. (A) unliganded KS1; (B) singly acylated KS1

obtained after treatment of DKS with propionyl-CoA either before

or after proteolysis; (C) singly acylated KS1 obtained after treatment

of DKS with n-butyryl-CoA either before or after proteolysis.



greatly reduced the number of digestion fragments and

resulted in the released domains being readily separable

by a reverse phase C4 column and monitored by

on-line UV and mass spectrometry. Our choice of con-

ditions for proteolysis was guided by earlier studies

in which protein products were identified using only

N-terminal sequencing. The cleavage patterns therefore

followed earlier precedents, but with some notable

exceptions. For example, it is particularly interesting

that for the first time KS1 and AT1 were released as

separate individual domains following tryptic digestion

of both DEBS1-TE and DKS. This observation will

help further studies on these two key domains.

Chromatographic separation of fragments released by

proteolysis makes quantification much easier. For exam-

ple, singly and doubly acylated loading module can be

separated and the relative amounts of the two forms

determined. The peak area of either UV absorbance or

mass spectrometric intensity can be used for quantifica-

tion. Even though the different species may coelute, the

relative ratios can be obtained from the deconvoluted

spectrum. During the deconvolution process, every

charge state of a particular species in the spectrum is

considered to determine the relative abundance.

The identity of each fragment was obtained by mass

mapping from the known amino acid sequence. Given

the good mass accuracy (0.01%), for any particular

observed mass a match could normally be found with

a unique tryptic fragment from the amino acid

sequence. In combination with N-terminal analysis,

this allowed us unambiguously to assign the identity of

each released domain fragment, and to assign both its

N- and C-terminus. The precise knowledge of bound-

aries of domains defined by proteolysis has proved

vital in the design of experiments to swap domains

between PKS assembly lines, and it also greatly facili-

tates the cloning of individual domain components for

catalytic and structural studies.

In our work, ion trap mass spectrometry was used.

Compared with the Fourier transform-ion cyclotron

resonance (FT-ICR) mass spectrometer [16,17], the ion

trap is less expensive and more widely available. In

addition, its coupling with HPLC is less complicated

and more widely established. Even though the ion trap

is a low-resolution instrument, it is more than ade-

quate for most of the analytical problems likely to be

posed by proteolysis studies of modular polyketides

and polypeptides. Masses can be reliably and routinely

measured with a precision of 0.01% for strong, well-

resolved peaks. Even with weak or overlapping peaks

the precision of mass measurements is sufficiently good

to allow differentiation of ligands differing by 14 Da,

the mass of a methylene group. The protocols are

therefore suitable for reliable identification of sites of

C-terminal cleavage or the likely chemical composition

of an attached ligand.

Although the main purpose of the present work was

to assess the utility of ion trap mass spectrometry for

analysis of the protein fragments produced by limited

proteolysis of modular PKSs, a number of significant

conclusions can already be drawn concerning the struc-

ture and function of the DEBS proteins.

Comparison of the proteolysis patterns found for

DEBS1-TE and DKS shows that module 1 can be

readily digested using trypsin while module 2 is more

resistant to proteolysis. Careful investigation of the

amino acid sequence of DEBS1-TE reveals that there

is no available trypsin cleavage site in the linker region

between ACP1 and KS2, which might account for the

observation that ACP1 was left as part of the large

fragment ACP1-KS2-AT2-KR2. However, several

potential cleavage sites do exist in the linker regions

between KS2 and AT2 and between AT2 and KR2.

DEBS3 module 6 also seems to be more resistant to

proteolysis than module 5. Further studies would need

to be carried out employing a wider variety of proteas-

es and different type I PKSs, to decide whether C-ter-

minal modules are always more resistant. Nevertheless,

the proteolysis results do seem to suggest that,

although the primary domain organization between

modules is very similar, the compactness of their qua-

ternary organization may be significantly different.

No significant difference in the digestion patterns

was observed between DEBS1-TE obtained in this

study and DEBS1 reported previously [13], which does

not contain a C-terminal TE domain. The digestion

pattern for the DKS was also similar to that of mod-

ule 1 from DEBS1-TE as previously suggested [13].

The similarity in digestion pattern between these three

proteins suggests that re-positioning of the TE domain

at the end of module does not significantly alter the

structure of the adjacent module.

Post-translational modification of acyl carrier protein

domains (ACPs) of a PKS by the attachment of a

4¢-phosphopantetheinyl moiety to a unique serine resi-

due in each ACP is a prerequisite for PKS activity. We

have shown here that limited proteolysis linked to

LC-MS analysis provides a quick and unambiguous way

of checking whether and to what extent this modifica-

tion occurs. DEBS1-TE and DKS, which were expressed

in their native host S. erythraea, showed 100% holo

form, as observed for both the loading ACP (ACPL)

and the first extension ACP (ACP1); while DEBS3,

which was heterologously expressed in E. coli, was fully

in the apo form as observed for the ACP of module 5

(ACP5). These observations provide direct evidence that



E. coli is incapable of post-translationally modifying

ACP with phosphopantetheine [19]. Recently, we have

characterized a 4¢-phosphopantetheinyl transferase fromS. erythraea which acts on DEBS, using the MS-based

approach presented here [22].

Because the proteolytic map is reproducible, once

conditions have been optimized, enzyme-catalyzed acy-

lation on individual domains can be probed directly by

the resulting mass increase. As every domain from the

DKS has been released either as an individual domain

or as a pair of domains, DKS was chosen for acylation

studies. It has been previously shown that the DEBS

loading module can be specifically labelled after incuba-

tion with radiolabelled propionyl-CoA and some other

unnatural CoA thioesters [13,21]. DEBS1-TE catalyzed

synthesis of triketide lactones in vitro revealed that the

DEBS loading module has a relaxed specificity for the

starter unit [20]. It has been proposed that two active

sites exist in the DEBS loading module: one is the serine

of the ATL domain; the other is the thiol of phospho-

pantetheine prosthetic group of the ACPL. The priming

of the PKS with the propionate starter is accomplished

through the ATL first recruiting the propionyl from pro-

pionyl-CoA, then the propionyl group being transferred

to the phosphopantetheine ‘swinging arm’. To obtain

direct and detailed information on starter loading and

transfer along the enzymatic assembly line of DEBS, we

individually incubated either the natural substrate pro-

pionyl-CoA, or the alternative non-natural substrate

n-butyryl-CoA, with the intact DKS, followed by a

rapid tryptic digestion and analysis using LC-MS. In

both cases, the resulting digestion patterns were the

same as those obtained from the unacylated protein,

showing that substrate attachment does not alter the

accessibility of the linker regions in the multienzyme.

Acylation of the loading module by either propionyl- or

n-butyryl-CoA was clearly observed after 10-min incu-

bation, with a maximum of two acyl groups being

attached to the loading module. This agrees with the

prediction of two active sites present in the loading mod-

ule. However, not all of the loading didomain was fully

acylated even after prolonged incubation (up to 1 h,

data not shown). In all cases, two types of acylated load-

ing module, mono (LM1) and double (LM2) acylation

were observed. The two forms were chromatographi-

cally separated, and the relative amount could therefore

be quantified by the corresponding peak area. The ratio

of the two forms varied depending on the substrate used,

as demonstrated here by the propionyl-CoA and n-buty-

ryl-CoA experiments. When n-butyryl-CoA was used, a

higher ratio of LM2 to LM1 was observed (Fig. 5B,C).

The LM1 peak from both substrates had a very similar

retention time to that of the free loading module, but

the LM2 had almost one and a half minute longer

retention time compared to the free form, and it

increased as the carbon number increased from three to

four as from propionyl to butyryl (Fig. 5A–C). The

chromatographic results seem to suggest that for

the doubly acylated loading module, at least one of the

attached acyl groups is exposed, but for the singly acyl-

ated loading module, under the chromatographic condi-

tions used, the acyl group is buried.

Our results show that when priming of DEBS occurs

with either propionyl or n-butyryl units, the KS1

domain is efficiently acylated as well as the loading

didomain. In addition, the observation of acyl-inter-

mediates on ATL-ACPL and KS1 demonstrates that

the formed acyl-enzyme intermediates are stable under

the digestion and analytical conditions used here, and

the ion trap mass spectrometry used is capable of ana-

lyzing the formed acyl intermediates with the ability to

distinguish propionyl or butyryl modification on a pro-

tein of over 60 kDa. Our observations, for the first

time, provide direct proof of the proposed mechanism

for priming, and further clearly show that all the three

active sites, ATL, ACPL and KS1, can be simulta-

neously primed by the natural substrate, propionyl-

CoA, and unnatural substrate, n-butyryl-CoA, in a

single multidomain enzyme.

To test whether the proteolytically released domains

retain activity, propionyl-CoA and n-butyryl-CoA were

also incubated individually with the digested DKS.

The loading didomain followed the same acylation pat-

terns as those with intact protein, providing evidence

that domains of type I PKS retain their intrinsic activ-

ity after cleavage of their linkers. It is significant that

KS1 was still observed fully acylated after acylation of

digested protein. The explanation for this may be KS

self-acylation, which was previously proposed for the

DEBS [23,24]. The other possibility is that within the

digestion mixture, the KS acylation occurs by in trans

collaboration with the released loading didomain. Such

in trans collaboration between functional domains has

been observed in other multidomain systems [12].

Further experiments using the thiol-directed reagent

NPDS to treat the released loading module, provide

evidence that the phosphopantetheine prosthetic group

is involved in the loading of the starter unit. Interest-

ingly, the experiments revealed that an intramolecular

disulfide bond was formed within the loading dido-

main when digested DKS was treated with NPDS. The

internal disulfide bond was also formed when intact

DKS was treated with NPDS (data not shown). When

checking the amino acid sequence of the released

DEBS loading module, three cysteine residues (C26,

C139 and C206) were found, and they all reside in the



ATL domain. Although the identity of the cysteine

residue involved remains to be established, the forma-

tion of this internal disulfide bond indicates that the

4¢-phosphopantetheinyl ‘swinging arm’ on the ACPL

can readily approach the ATL domain.

In conclusion, our strategy for limited proteolysis in

combination with on-line liquid chromatography ion

trap mass spectrometry studies on the multifunctional

proteins, DEBS1-TE, DEBS3 and DKS as model sys-

tems, introduces an additional powerful experimental

tool for studies on the substrate specificity and organiza-

tion of type I PKSs. The methodology is based on

accessible and simple mass spectrometry equipment

rather than advanced FT-ICR-MS. Nevertheless, the

technology can achieve mass accuracy within 0.01% for

proteins up to 79 kDa and so can distinguish between

ligands which differ by as little as a methylene or an oxy-

gen. For chain initiation on DEBS, we have directly

demonstrated that multiple sites can be simultaneously

loaded with propionate or other starter acid units. This

raises the possibility that most sites on a longer assembly

line are operating simultaneously on different growing

chains. We have also discovered different degrees of sus-

ceptibility to proteolysis from one module to another,

which may reflect differential tightness of packing of

domains in the module. The technology appears appro-

priate for direct domain-by-domain investigation of

intermediates in the chain extension process on type I

modular PKS proteins. The information provided by

such studies should be particularly useful in optimizing

the efficiency of engineered PKS multienzymes.

Experimental procedures

General

Three proteins were used in this study: two bimodular pro-

teins, DEBS1-TE and DEBS3, and one unimodular protein

DKS. DEBS1-TE and DKS were expressed in S. erythraea

and purified as described previously [6,7]. DEBS3 was

heterologously expressed in E. coli, and purified as pre-

viously described [22]. Propionyl-CoA, n-butyryl-CoA and

l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-

treated trypsin were purchased from Sigma (Poole, Dorset,

UK). 4-Nitrophenyl disulfide (NPDS) was purchased from

Aldrich (Poole, Dorset, UK).

Liquid chromatography-mass spectrometry

(LC-MS) analysis

Analysis was carried out on a HP 1100 (Hewlett-Packard,

Wilmington, DE, USA) liquid chromatography coupled

with an LCQ Classic (ThermoFinnigan, San Jose, USA)

mass spectrometer fitted with an electrospray-ionization

source. After proteolysis, the digestion mixture was applied

to a reverse phase column (Vydac, Protein C4,

4.6 · 250 mm, 300 A) and eluted with a linear gradient

from 35% to 55% acetonitrile (0.1% trifluoroacetic acid) ⁄water (0.1% trifluoroacetic acid) over 40 min at a flow rate

of 0.7 mLÆmin)1. The eluate was monitored by a diode array

detector selecting the UV absorbance at 214 and 280 nm.

The flow of eluate was split prior to the mass spectrometer.

Diverted eluate was collected manually and fractions were

N-terminally sequenced using an automated sequencer

(ABI, Foster City, CA, USA). The spray voltage of the

mass spectrometer was 4.5 kV, and the capillary tempera-

ture was 200 �C. All data were acquired in the positive-ion

mode at unit mass resolving power between m ⁄ z 600 to m ⁄ z2000. The LC-MS system was controlled by the xcalibur

software (version 1.1, ThermoFinnigan, San Jose, CA,

USA). The mass spectrometric data were processed and

transformed using the bioworks software (version 1.1,

ThermoFinnigan). The centroids of the ‘deconvoluted’

peaks were used to assign the observed masses. The software

paws (Proteometrics, NY, USA) was used to search the

tryptic fragments based on the known amino acid sequence.

Limited tryptic-digestion of DEBS1-TE, DEBS3

and DKS

Purified DEBS1-TE was incubated with TPCK-treated tryp-

sin at varying protein ⁄ trypsin ratios of 500 : 1, 200 : 1,

100 : 1 and 75 : 1 (w ⁄w) in digestion buffer (400 mm potas-

sium phosphate, pH 7.4, 1 mm EDTA, 1 mm dithiothreitol,

20% glycerol). Reactions were carried out at 30 �C for dif-

ferent times of incubation. Purified DEBS3 was incubated

with TPCK-treated trypsin at a protein ⁄ trypsin ratio of

either 250 : 1 or 75 : 1 (w ⁄w) in digestion buffer. Reactions

were carried out at 30 �C for 5 min. Purified DKS was incu-

bated with TPCK-treated trypsin at a protein ⁄ trypsin ratio

of 800 : 1 (w ⁄w) in digestion buffer. Reactions were carried

out at 30 �C for 5, 15, 30, and 60 min to determine the opti-

mal digestion conditions under which stable proteolytic

fragments were released. Digestion of DKS was also carried

out at a protein ⁄ trypsin ratio of 80 : 1 (w ⁄w) at 30 �C for

5 min. All the digestions were terminated by loading the

digestion mixture directly onto the pre-equilibrated (35%

acetonitrile ⁄ 0.1% trifluoroacetic acid) C4 column.

Propionyl-CoA/n-butyryl-CoA incubation with

intact and digested DKS

Intact DKS

Guided by previous measurements of substrate concentra-

tion required for saturation of the loading didomain [21],

purified DKS (6 lm) was incubated individually with each

acyl-CoA (6 mm) 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 reaction was carried

out at 30 �C for 10 min. To minimize subsequent chemical

hydrolysis of the acyl-enzyme intermediates formed, trypsin

was added immediately to the reaction mixture at a pro-

tein ⁄ trypsin ratio of 80 : 1 (w ⁄w), and digestion was per-

formed at 30 �C for 5 min, before analysis by LC-MS.

Proteolysed DKS

Six micromolar DKS, proteolysed with trypsin as above,

was incubated with 6 mm propionyl-CoA in a total volume

of 30 lL, containing 400 mm potassium phosphate

(pH 7.4), 1 mm EDTA, 1 mm dithiothreitol, and 20% gly-

cerol. Incubations were at 30 �C for 5, 10 or 20 min,

respectively. After incubation, the reaction mixture was ana-

lyzed directly by LC-MS. Similarly, n-butyryl-CoA was also

incubated with proteolysed DKS, and analyzed by LC-MS.

Probing the sites of acylation of loading

didomain with 4-nitrophenyl disulfide

Purified DKS was subjected to tryptic proteolysis as des-

cribed above. After limited proteolysis, 4-nitrophenyl disul-

fide (NPDS) was added to the trypsin-digested DKS (6 lm)to a final concentration of 52 lm. The reaction mixture was

incubated at 30 �C for 5 min, and a portion was then ana-

lyzed by LC-MS. To the rest of the reaction mixture pro-

pionyl-CoA was added to a final concentration of 6 mm,

and after incubation at 30 �C for a further 10 min the mix-

ture was analyzed by LC-MS. To test whether NPDS had

induced the formation of an intramolecular disulfide bond

within the loading didomain (ATL-ACPL), after incubating

NPDS with the digested DKS, a large excess of dithiothrei-

tol was added to the reaction mixture, and left at room

temperature for 10 min. Excess dithiothreitol was then

removed from the peptide mixture by buffer exchange using

a CentriprepTM concentrator (10 kDa cut-off) (Amicon,

Bedford, MA, USA). Propionyl-CoA was added to the

mixture, and the reaction was performed at 30 �C for

10 min, and the product mixture then analyzed by LC-MS.

Acknowledgements

We gratefully acknowledge the financial support of the

Biotechnology and Biological Sciences Research Coun-

cil (UK) for this work through a project grant to PFL

and JS. JGB thanks the EU for a Marie Curie Post-

doctoral Research Fellowship.

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