Staunton, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK Fax: +44 1223 762018 Tel: +44 1223 766041 E-mail: js24@cam.ac.uk Received 10 November 20
Trang 1revealed by limited proteolysis and ion-trap mass
spectrometry
Hui 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: js24@cam.ac.uk
(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.
Trang 2multienzyme polypeptides essentially in the order that
they are used
Modular PKSs are clearly amenable to rational
gen-etic manipulation of the biosynthgen-etic 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.
Trang 3Multifunctional 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 30C, 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⁄ trypsin ratio in the range from 50 : 1 to 100 : 1 (w⁄ w) at 30 C for 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 C for 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.
Trang 4other 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-ACPLseemed
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¢-phosphopantetheinyl prosthetic group of both the loading and extender ACPs was confirmed by the fact that the observed mass of
ATL-ACPLand 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 sequence a Observed mass (Da) b Expected mass (Da)
40594 (holo)
56006 (holo)
56392 (holo)
150168 (holo)
a
All cleavages were at C-terminal of R residues (K is absent from the linker regions).bThe error bars reported are based on at least three independent experiments *Signifies an unidentified residue.
Trang 5tion pattern was the same in both cases The LC trace
obtained for the digestion mixture at a protein⁄ trypsin
ratio 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.
Fragment identity Corresponding sequence N-Terminal sequence a Observed mass (Da) b Expected mass (Da)
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.
Trang 6released 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
30C 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 -ACPLand 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.
Fragment identity Corresponding sequence N-Terminal sequence a Observed mass (Da) b Expected mass (Da)
39 510 (holo)
56 006 (holo)
56 392 (holo)
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.
Trang 7Digested 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 30C 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 AT L -ACP L ; LM1 and LM2, signify singly and doubly
acylat-ed loading module, respectively; LM(S-S), loading module containing an internal disulfide bond between the ATLand the phosphopantetheine
of ACPL; LM1(S-S), singly acylated loading module containing an internal disulfide bond between the ATLand the phosphapantetheine of ACP L It is not known whether the single acyl group is attached exclusively to the active site of AT L or of ACP L , or both.
Trang 8Probing 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 30C 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 containelut-ed 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 AT L -ACP L (A) Mass spectrum of the loading didomain
ATL-ACPLresulting from tryptic digestion of DKS; (B) mass spectrum of the loading didomain ATL-ACPLresulting from tryptic digestion of DKS, after subsequent treat-ment with NPDS The formation of an inter-nal disulfide bond between the ATLand ACPL, induced by NPDS treatment, results
in alteration of the m ⁄ z distribution to a higher mass range (see text for details).
Trang 9for 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 ACPLwas blocked by the treatment
Fig 5 Deconvoluted mass spectra of loading didomain ATL-ACPLreleased 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.
Trang 10of 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.