Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry Hui Hong 1 , Antony N. Appleyard 2 , Alexandros P. Siskos 2 , Jose Garcia-Bernardo 2 , James Staunton 1 and Peter F. Leadlay 2 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 (AT L -ACP L ), 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 ⁄ 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 AT L -ACP L ; 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 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 2375 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 AT L -ACP L . 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 AT L -ACP L 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¢-phosphopantetheinyl prosthetic group of both the loading and extender ACPs was confirmed by the fact that the observed mass of AT L -ACP L 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 sequence a 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 AT L -ACP L 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. Limited proteolysis and MS of modular PKSs H. Hong et al. 2376 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS tion 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 AT L -ACP L , 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 AT L -ACP L 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) 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. H. Hong et al. Limited proteolysis and MS of modular PKSs FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 2377 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 AT L -ACP L 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 AT L -ACP L 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 AT L - ACP L 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 AT L - ACP L 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) 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 AT L -ACP L 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. Limited proteolysis and MS of modular PKSs H. Hong et al. 2378 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 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 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 AT L and the phosphopantetheine of ACP L ; LM1(S-S), singly acylated loading module containing an internal disulfide bond between the AT L and the phosphapantetheine of ACP L . àIt is not known whether the single acyl group is attached exclusively to the active site of AT L or of ACP L , or both. H. Hong et al. Limited proteolysis and MS of modular PKSs FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 2379 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 AT L -ACP L . (A) Mass spectrum of the loading didomain AT L -ACP L resulting from tryptic digestion of DKS; (B) mass spectrum of the loading didomain AT L -ACP L resulting from tryptic digestion of DKS, after subsequent treat- ment with NPDS. The formation of an inter- nal disulfide bond between the AT L and ACP L , induced by NPDS treatment, results in alteration of the m ⁄ z distribution to a higher mass range (see text for details). Limited proteolysis and MS of modular PKSs H. Hong et al. 2380 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS for a disulfide bond-linked dimer of AT L -ACP L . 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 ACP L was blocked by the treatment Fig. 5. Deconvoluted mass spectra of loading didomain AT L -ACP L 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. H. Hong et al. Limited proteolysis and MS of modular PKSs FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS 2381 of NPDS, and only the active site serine residue of AT L 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 ACP L 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 [ 14 C]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 AT L [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. Limited proteolysis and MS of modular PKSs H. Hong et al. 2382 FEBS Journal 272 (2005) 2373–2387 ª 2005 FEBS [...]... domain Although the identity of the cysteine residue involved remains to be established, the formation 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,... protein, providing evidence that domains of type I PKS retain their intrinsic activity 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... multienzyme complex of Bacillus stearothermophilus and their role in catalysis Eur J Biochem 267, 7158– 7169 11 Bantscheff M, Weiss V & Glocker MO (1999) Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry Biochemistry 38, 11012–11020 12 Hijarrubia MJ, Aparicio JF & Martin... from one module to another, which may reflect differential tightness of packing of domains in the module The technology appears appropriate 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... 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 concentration... extension module of the erythromycin polyketide synthase Biochemistry 41, 2719–2726 7 Bycroft M, Weissman KJ, Staunton J & Leadlay PF (2000) Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase Eur J Biochem 267, 520–526 8 Mally MI, Grayson DR & Evans DR (1981) Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis... differ by as little as a methylene or an oxygen 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 susceptibility to proteolysis from... 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 adequate for most of the analytical problems likely to be posed by proteolysis studies of modular polyketides and polypeptides Masses... (2003) Domain structure characterization of the multifunctional alpha- aminoadipate reductase from Penicillium chrysogenum by limited proteolysis – activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain J Biol Chem 278, 8250–8256 13 Aparicio JF, Caffrey P, Marsden AFA, Staunton J & Leadlay PF (1994) Limited proteolysis and active-site studies of 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 analyzing the formed acyl intermediates with the ability to distinguish propionyl or butyryl modification on a protein of over 60 kDa Our observations, for the first time, provide direct proof of the proposed mechanism for priming, and further clearly show . Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry Hui Hong 1 , Antony N. Appleyard 2 ,. AT L [21]. Discussion DEBS1-TE, DEBS3 and DKS were subjected to limited tryptic digestion, and the digestion conditions were optimized for each protein so that