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

The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry Hui Hong1,2, Peter F Leadlay2 and James Staunton1 Department of Chemistry, University of Cambridge, UK Department of Biochemistry, University of Cambridge, UK Keywords enzyme-bound intermediate; erythromycin; limited proteolysis; liquid chromatographymass spectrometry; polyketide synthase Correspondence H Hong, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK *Fax: +44 1223 766002 Tel: +44 1223 333659 †E-mail: hh230@cam.ac.uk J Staunton, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK Fax: +44 1223 336362 Tel: +44 1223 336300 E-mail: js24@cam.ac.uk (Received 28 July 2009, revised 29 September 2009, accepted 30 September 2009) doi:10.1111/j.1742-4658.2009.07418.x A catalytically competent, homodimeric diketide synthase comprising the first extension module of the erythromycin polyketide synthase was analysed using MS, after limited proteolysis to release functional domains, to determine the pattern of covalent attachment of substrates and intermediates to active sites during catalysis Using the natural substrates, the acyltransferase and acylcarrier protein of the loading module were found to be heavily loaded with propionyl starter groups, while the ketosynthase was fully propionylated The acylcarrier protein of the extension module was partly occupied by the product diketide, and the adjacent chain-releasing thioesterase domain was vacant, implying that the rate- limiting step is transfer of the diketide from the acylcarrier protein to the thioesterase domain The data suggest an attractive model for preventing iterative chain extension by efficient repriming of the ketosynthase domain after condensation Use of the alternative starter unit valeryl-CoA produced an altered pattern, in which a significant proportion of the extension acylcarrier protein was loaded with methylmalonate, not diketide, consistent with the condensation step having become an additional slow step Strikingly, when NADPH was omitted, the extension acylcarrier protein contained methylmalonate and none of the expected keto diketide, in contrast to results obtained previously by mixing individual recombinant domains, showing the importance of also studying intact modules The detailed patterns of loading of the extension acylcarrier protein (of which there are two in the homodimer) also provided the first evidence for simultaneous loading of both acylcarrier proteins and for the coordination of timing between the two active centres for chain extension Introduction Polyketides are a large and diverse group of secondary metabolites that are produced by a common biosynthetic strategy in bacteria, fungi, plants and animals The term polyketide refers to the early steps of a typical pathway, in which a starter acyl residue is extended by successive addition of acyl residues, each equivalent to Abbreviations ACP, acyl carrier protein; AT, acyl transferase; DEBS, 6-deoxyerythronolide B synthase; DKS, diketide synthase; FAS, fatty acid synthase; KR, ketoreductase; KS, ketosynthase; PKS, polyketide synthase; SNAC, thioester of N-acetylcysteamine; TE, thioesterase * [Correction added on November 2009 after first online publication: The fax number is wrong, it should be 766002, not 966002] † [Correction added on November 2009 after first online publication: the email address for the first corresponding author is wrong, it should be hh230@cam.ac.uk, not js24@cam.ac.uk] FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7057 Changing patterns of covalent active-site occupancy H Hong et al the general structural ketene unit, RCH=C=O, until the linear chain of carbons reaches the desired length Subsequent diverse biosynthetic transformations generate an enormously varied set of structures [1,2] The catalytic enzymes responsible for the chain-extension processes show a remarkable degree of structural and mechanistic homology across the wide range of biosynthetic organisms There are large differences, however, in the manner in which the enzymes are housed in multienzyme clusters At one extreme, a single set of chain-extension enzymes carries out all the chain-extension steps (iterative operation); at the other extreme, there are systems which have a separate enzyme for every step of the chain-extension processes (modular assembly line operation) A further source of variance is found in the nature of association between enzymes in the clusters; in some systems (Type II) the individual enzymes are readily dissociable; others (Type I) contain large assemblies of covalently linked catalytic sites The work in this investigation applies exclusively to Type I modular systems that occur largely in bacteria and produce so-called ‘complex’ polyketides These polyketides are a very large and diverse group of secLoad Module Module Module DEBS1 ER DEBS2 SH S ACP KS AT ACP TE SH SH S S O O O OH OH OH OH S S O OH O KS AT SH S S KR KR KS AT ACP KS AT ACP SH SH Module DEBS3 KR DH AT ACP KS AT ACP KS AT ACP Module Module KR KR ondary metabolites, produced largely (if not exclusively) by certain genera of bacteria They include some of the most valuable natural products to have reached the clinic, such as the antibacterial erythromycin A, the antiparasitic avermectin and the immunosuppressant rapamycin [1] They are produced on giant modular multienzyme assembly lines [polyketide synthases (PKSs)] in a linear chain-building process akin to that of fatty acid biosynthesis, with the chief difference being that PKSs may recruit a far greater variety of starter units and extender units, while the overall length of the product and the level of reduction of each unit, as it is incorporated, may also vary [2–5] The specificity is assured by utilizing a different module of fatty acid synthase (FAS)-related activities for each cycle of chain extension, as illustrated in Fig for the erythromycin-producing PKS [6-deoxyerythronolide B synthase (DEBS)], which catalyzes assembly of the aglycone 6-deoxyerythronolide B from one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA [2,5–8] Figure also shows the arrangement of enzymatic domains in an engineered diketide synthase (DKS) containing only one extension O O O OH OH O Me Me O OH Cyclise on TE domain OH O OH OH O OH OH Me Me Load OH OH Me Intermediates Module Me O O OH OH 6-Deoxyerythronolide B (6-DEB) Me DKS OH KR Me AT ACP KS AT ACP TE OH OH SH OH S S Hydrolyse on TE domain HO O O O OH Me OH Me Me Diketide acid Fig PKS assembly line responsible for assembling the macrolide core of 6-deoxyerythronolide B (6-DEB), as revealed by sequencing the genes Each cycle of chain assembly is carried out by a dedicated set of enzymes so that there is a separate enzyme for every step The enzymes are organized in sets (modules), one for each cycle Each module has the correct set of enzymes for the extent of keto group modification (hydroxyl, enoyl, saturated methylene, colour coded), which ensures that the fatty acid chain-extension cycle is appropriately truncated and the correct transfer path is followed At the terminus there is a TE domain that carries out cyclisation and release of the first enzyme-free product, 6-DEB, as a macrolactone The truncated model DKS is shown below It carries out the operations of module and then releases the diketide product as a fatty acid 7058 FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS H Hong et al module [9], from which the diketide product is released by the action of the C-terminal thioesterase (TE) The purified DKS, generated from DEBS by deleting extension modules 2–6, has been shown to hydrolyze and release the predicted diketide product in vitro [9], albeit with a turnover that is low compared with the betterstudied triketide synthase, DEBS1-TE, where the TE can operate its preferred mode of chain release via cyclization [10] At first sight, the modular paradigm for enzymatic catalysis in these modular PKSs, one of which is shared with nonribosomal peptide synthetases [11], appears simple because of the apparently direct correspondence between the enzyme activities of a given module and the chemical structure produced during that cycle of extension However, despite extensive study, it is still not understood how these giant enzymatic assemblies are controlled and orchestrated In fact, under certain conditions, modular PKSs have been shown to give aberrant products in vivo in which individual enzymecatalyzed steps [12], or even whole modules, are ‘skipped’ [13,14], while in other cases extension modules operate more than once (iteration or ‘stuttering’) [15–17] In a few examples, such skipping or iteration is actually required in order to produce the natural product [18–21] Recent structural studies on the intact animal FAS multienzyme [22,23] and on modular PKS domains [24–27] have also given a fresh impetus to the question of control and orchestration of the individual steps involved in chain extension The work on FAS has revealed a high mobility of certain domains and potentially a key role for major conformational changes during catalysis [22,23] Both animal FAS and modular PKS are functional homodimers, which raises additional questions about the interactions between the active sites of an identical pair of modules We report here the use of ion-trap MS and the DKS model system [9] to study the identity of multienzymebound intermediates and to establish the pattern and level of covalent attachment of substrates and intermediates to individual active sites, during catalysis in vitro on a modular PKS For dissociated (Type II) FAS and PKS, monitoring the nature of acyl chains attached to the acylcarrier protein (ACP) followed quickly upon the introduction of electrospray MS [28,29] and it continues to give valuable mechanistic insights [30,31] into such systems Unfortunately the sheer size of modular PKS multienzymes has hampered their analysis in this way One successful approach [32,33] was to degrade the multienzymes to short peptide fragments, fractionate the complex mixture and use high-resolution electrospray ionization Fourier-Transform mass spectrometry Changing patterns of covalent active-site occupancy (ESI ⁄ FTMS) to pick out the active-site peptides and determine the nature of the covalently attached group [34,35] In addition, specific ejection of the phosphopantetheinyl prosthetic group from the ACP can be induced in the mass spectrometer, allowing highly accurate determination of the mass of the attached species [35] A convenient ion-trap MS-based approach may also be used in which limited proteolysis [36] is used to generate domain-sized fragments for liquid chromatography (LC) ⁄ MS analysis We have previously validated this technology for the DKS (Fig 2A) [37] and used it to demonstrate, for the first time, that during the loading of the synthase with the natural starter unit propionyl-CoA (as well as from the alternative starter units acetyl-CoA, butyryl-CoA and valeryl-CoA), three different active sites in both DKS subunits become almost completely acylated, namely the acyltransferase (AT) and ACP domains of the loading module and the ketosynthase domain (KS1) (Fig 2B) Here, we have used the method to establish the pattern of covalent intermediates attached to various active sites of an intact PKS module, during catalysis of overall diketide formation This has revealed previously unsuspected features of chain elongation on such enzymes Results The same methodology as used in our previous study [37] was first applied to investigate the loading of the natural methylmalonyl extender unit, derived from methylmalonyl-CoA, onto the extension module ACP (ACP1) of DKS After incubation with commercial methylmalonyl-CoA for 10 min, the DKS protein was digested and subjected to analysis by HPLC ⁄ MS The extension module AT domain from DKS (AT1) appears as two fragments corresponding to alternative sites of proteolysis; one fragment has a molecular mass of 32582 Da and the second has a molecular mass of 32739 Da As expected, after incubation with methylmalonyl-CoA, both fragments showed two extra peaks at 32684 and 32839 Da, respectively, corresponding to the addition of a methylmalonyl moiety (see Fig 3) The ratios of the intensities of the peaks for the loaded and unloaded forms of each AT1 fragment were almost identical, at 60% and 40%, respectively (Table 1) Similarly, the fragment for the ACP-TE di-domain showed two peaks, one with a molecular mass of 39506 Da corresponding to the unloaded form and the other with a molecular mass of 39604 Da, corresponding to the form loaded with methylmalonate (see Fig 3) In this case, a ratio of approximately 55% loaded to 45% unloaded forms was observed (Table 1) FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7059 Changing patterns of covalent active-site occupancy H Hong et al A Control experiment; no incubation prior to analysis Load Module KR Proteolysis AT ACP AT ACP KS AT ACP TE OH SH OH OH AT KR ACP TE OH SH OH SH SH KS OH TE OH SH SH B After incubation with propionyl CoA Load Module KR Proteolysis AT ACP KS AT ACP TE O S O OH O AT ACP OH KS O S KR ACP TE OH OH S O SH AT O TE OH SH S O O C After incubation with purified methylmalonyl CoA Load Module KR Proteolysis AT ACP KS AT ACP TE OH SH O SH O HO2C OH AT ACP SH OH SH S O KS KR AT ACP TE OH O O CO2H TE OH S O CO2H CO2H Fig Results of proteolysis of the DKS followed by HPLC coupled to electrospray MS Experiments (A) and (B) were reported in a previous publication [37]; experiment (C) is part of the current results Surprisingly, analysis of the fragments derived from the loading module in this initial experiment produced evidence that both the AT and ACP domains of the loading module, and the KS domain, were loaded with propionate The purity of the commercial methylmalonate was therefore checked by HPLC, which revealed contamination with propionyl-CoA Although the level of contamination was low, it was sufficient to explain the unexpected propionate loading Purification by HPLC gave propionyl-CoA free of the methylmalonyl analogue Repeating the experiment gave the same results for loading of AT1 and ACP-TE di-domains, but the domains of the loading 7060 module and the KS were completely unloaded, as indicated in Fig 2C Apart from demonstrating the importance of using pure materials, the results with the pure methylmalonate were significant in removing any possibility that methylmalonate decarboxylation by the KS provides an alternative source of the propionate building block used in the first condensation step The DKS was also incubated with malonyl-CoA under the same conditions used for methylmalonylCoA No loaded residues were detected for any of the domains, showing that the AT1 domain is highly substrate specific, unlike the AT domain of the loading module (data not shown) FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS H Hong et al Changing patterns of covalent active-site occupancy AT ACP-TE Methylmalonyl adduct Methylmalonyl adducts Unloaded forms Fig The electrospray mass spectra produced by the AT and ACP-TE fractions derived from the extension module after incubation with methylmalonyl-CoA The AT domain shows two sets of peaks that arise from alternative sites of proteolysis in the downstream linker to KR 32 400 32 800 Unloaded form 33 200 39 400 39 500 39 600 Mass (Da) The DKS was then incubated with various combinations of substrates that should allow synthesis of a diketide product and then analyzed as before to determine the nature and extent of occupancy of the KS, AT and ACP chain-extension domains of module [the ketoreductase (KR) domain does not have a substrate covalently bound] and of the adjacent TE domain Various sets of incubation conditions, listed in Table 1, were explored The possible adducts on the various domains are shown in Fig First, the DKS was incubated with propionyl-CoA to supply the native starter unit, methylmalonyl-CoA as the source of extender unit, and, with NADPH, to carry out the keto-group reduction step catalysed by the KR domain After a sufficient incubation period to establish steady turnover (10 min), the mixture was analysed using the standard protocol to determine the extent of loading on the domains of module and its attached TE domain MS analysis of the KS1 fraction showed that the active site was fully loaded with propionate The absence of the free form of the KS shows that loading of propionate onto the KS via the loading module is 39 700 Mass (Da) not rate-limiting under these conditions The mass spectrum for the fraction containing the TE domain and the key ACP-TE domain is shown in Fig 5B Only one peak was observed for the TE domain, with a mass corresponding to the unloaded form From this it can be concluded that release of the diketide intermediate from this domain is faster than its acylation by diketide transfer from ACP1 Therefore any covalently attached species detected on the ACP-TE di-domain is resident only on the ACP1 thiol Two peaks are seen, one corresponding to the unloaded form and the other to the form loaded with diketide (see Table 1) From the increased mass this could have been the keto diketide form or the hydroxy diketide form, or a mixture of the two Surprisingly, given the results of incubation with methylmalonyl-CoA alone, there was no evidence for the methylmalonyl derivative of the chain-extension ACP, despite the presence of the free thiol form of the domain The nature of the diketide adduct in this experiment was determined by treatment of a sample of the loaded ACP-TE di-domain with hydrazine to remove the added diketide ligand as the hydrazide derivative Table Occupancy levels of intermediates on chain extension ACP1 under various assay conditions Percentages of derivatized forms of the chain-extension ACP domain Incubation mixtures Free thiol Methylmalonate Ketodiketide Hydroxydiketide 45 42 51 37 45 55 0 35 55 N⁄A N⁄A 0a 0a N⁄A N⁄A 58 49a 28a a Methylmalonyl-CoA Malonyl-CoA Propionyl-CoA; methylmalonyl-CoA; NADPH Butyryl-CoA; methylmalonyl-CoA; NADPH Valeryl–CoA; methylmalonyl-CoA; NADPH Propionyl-CoA; methylmalonyl-CoA; no NADPH Absence of keto-diketide assumed by analogy with experiment FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7061 Changing patterns of covalent active-site occupancy H Hong et al A KR AT KS ACP AT OH SH SH OH O S S TE ACP OH S SH B O R O R O O O O R CO 2H OH R C S S O CO 2H S O O O R OH R 28 000 Fig The range of biosynthetic intermediates predicted to be covalently bound to the various enzyme active sites in the course of a chain-extension cycle and product release on the DKS 32 000 36 000 40 000 Mass (Da) D Free 39 509 Methylmalonate 39 611 First, suitable conditions for the reaction were established by a control study with a synthetic sample of the N-acetylcysteamine analogue (Fig 6) The extract of the reaction mixture showed a product which was identified by high-resolution MS (calculated for expected product [M+H]+ 147.1133, found [M+H]+ 147.1150) Further support for the structure of the hydrazide was obtained from an MS ⁄ MS spectrum, which produced a fragment ion for loss of H2O at m ⁄ z 129.1 Repeating this experiment with the loaded ACPTE di-domain gave the same product, as judged by MS analysis Careful examination of the LC-MS trace failed to show evidence for any of the possible hydrazine derivatives of the keto analogue of the diketide (m ⁄ z 145 or 127), and so a confirmatory control experiment with the keto derivative was not necessary Analysis of the ACP-TE fraction recovered from the hydrazine treatment showed it to be in the free form, as expected These experiments confirmed that the DKS is active in diketide synthesis under these conditions, and that the hydroxy diketide intermediate accumulates on the synthase in a significant quantity It appears that very little, if any, of the accumulated diketide intermediate is in the unreduced keto-form When the natural starter unit was replaced with butyrate (an increase in size of 14 mass units), analysis of the DKS showed that the KS domain was fully loaded by butyrate As with propionate, the chainextension ACP (ACP1) showed a peak for the unloaded form and a peak for the form loaded with the heavier diketide analogue (Fig 5C) The two 7062 Diketide 39 644 39 300 39 500 39 700 39 900 Mass (Da) Fig MS results from experiments 3, and (A) Control experiment without added precursors (B) Incubation with propionate as the starter in experiment (C) Incubation with butyrate as the starter in experiment (D) Incubation with valerate as the starter in experiment 5, showing the expanded version of the ACP-TE region species were now present in approximately equal amounts (Table 1) Again, the isolated TE domain was free of diketide derivative, and there was no evidence for the methylmalonyl derivative of the ACP Incubation of DKS with valeryl-CoA likewise led to the complete loading of KS with the valeryl group, but here there was a marked change in the pattern of loading of ACP1 This now showed three peaks: free thiol group (37%), the valeryl diketide derivative (28%) and, in addition, the methylmalonyl derivative (35%) (Fig 5D) not seen when either propionyl-CoA or butyryl-CoA was used (Table 1) As in the previous two experiments, the TE was not found to be acylated FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS H Hong et al Changing patterns of covalent active-site occupancy ACP TE OH SNAC O H2NHN NH2NH2 OH O OH NH2NH2 S hydrolase activity [38], which may also influence the steady-state level of acylation of AT1 and ACP1 domains O OH Fig Chemical treatment of the diketide N-acetylcysteamine (NAC) derivative and the ACP-TE derivative with hydrazine to release the hydrazide product for analysis by MS SNAC, thioester of N-acetylcysteamine Finally, an experiment was carried out in which the natural substrates propionyl-CoA and methylmalonylCoA were supplied, but in which NADPH was not supplied The aim was to see if the keto-ester intermediate accumulated, and, if so, which stereoisomer dominated in the keto-ester product As expected, the KS domain was found to be fully loaded with a propionyl unit, and the two domains of the loading module were also substantially loaded with the starter acyl residue units However, the omission of NADPH had a marked effect on the pattern of loading on the chainextension ACP (ACP1) in this experiment There was a peak for the methylmalonyl derivative, as well as for the free thiol form, but there was no detectable peak for any diketide intermediate Interestingly, the relative proportions of the free thiol form of ACP1 (45%) and the methylmalonyl form (55%) were identical to those observed in the experiment in which no starter unit was supplied, and the TE domain was again unloaded Discussion Incubation of DKS with methylmalonyl-CoA gives incomplete acylation of AT1 and ACP1 domains In our previous experiments [37] we showed that incubation of the DKS in vitro with saturating concentrations of starter substrates led to complete acylation of the KS domain and nearly complete acylation of both domains in the loading module By contrast, in the present study we found that the level of loading of methylmalonate on both the chain-extension AT and ACP domains was considerably less than 100% As the AT-catalyzed reaction is readily reversible, the extent of loading of methylmalonate on the AT and ACP domains is probably determined (at least in part) by the relative stabilities of free methylmalonyl-CoA ester and the loaded forms of the domains, and thus by the concentrations of methylmalonyl-CoA and protein used in this study We have also previously demonstrated that the AT domains of purified DEBS possess a slow methylmalonyl-CoA Identification of rate-limiting steps, and a model for suppression of iteration and the maintenance of fidelity of reduction In the presence of all the (natural) substrates required for diketide synthesis on the DKS, the relatively high level of loading of multiple sites (> 50%) persists, apart from the TE domain It would appear that under these conditions, two chains can be elongated at the same time, and that the rate-limiting step is the transfer of the diketide intermediate from the chain-extension ACP to the TE, not the subsequent release of the diketide acid from the TE This bottleneck at the exit stage causes a backlog of intermediates to build up at previous steps When the alternative (progressively poorer) starter substrates butyryl-CoA and valeryl-CoA were used, the KS remained fully loaded and the extent of methylmalonate loading on the chain-extension AT was not significantly changed By contrast, there were dramatic changes in ACP1 occupancy With butyrate, the proportion of ACP1 loaded with diketide fell significantly, consistent with a slowing of the rate of the condensation step relative to the offloading step (we assumed that all the diketide intermediate was in the hydroxy form in experiments and 5) In the valerate experiment there was a more dramatic change The proportion of the diketide intermediate fell even further (below 50%) and a detectable amount of ACP1 was found to be loaded with methylmalonate It would appear that the condensation step, under these conditions, provides a significant additional bottleneck in the chain-assembly process However, the key point to emerge from these experiments is that by using the normal substrates, all the active sites are found to be heavily loaded This situation, if it holds for PKSs in vivo, would contrast sharply with a conventional metabolic pathway where overall rate control is dominated by early enzymes to avoid the accumulation of large pools of enzyme-free intermediates Regulation of productivity at the release step could also provide important advantages for the control of fidelity in natural modular PKS assembly lines In the case of the complete DEBS assembly line, for example, regulation at the stage of product release would cause all the ACP and KS domains to be loaded by appropriate intermediates and all the AT domains to be primed with the relevant building blocks This would FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7063 Changing patterns of covalent active-site occupancy H Hong et al lead to an intermittent mode of operation, in which successive rounds of chain-extension cycles would be triggered by release of the heptaketide intermediate from the ACP domain in the last module (Fig 1), rather like the operation of an automatic drinks-can dispenser In each module, when the KS domain has become free, it would be immediately reloaded with substrate from the upstream ACP This would prevent back-transfer of biosynthetic intermediates subsequently generated on the ACP domain within that module, and suppress iterative use of the module The term ‘congestion control’ has been suggested for this effect [5] It is consistent with this hypothesis that aberrant iteration in a PKS has been seen when the levels of a PKS were increased without also increasing the levels of intracellular precursors [15] The throttling back of the process of product release could also contribute to the maintenance of fidelity in the reductive steps of the DEBS synthetic operations It is vital that in every cycle all the programmed reductive steps are completed before downstream transfer of the fully modified intermediate from the ACP to the KS domain of the next module Molecular recognition cannot be the sole factor in this, as shown most clearly for the mycolactone PKS, where all 16 extension KS domains have an essentially identical sequence [39] and cannot therefore be expected to discriminate between the various intermediates sequentially generated in the upstream module An alternative and simpler mechanism for the control of fidelity is that all the steps of keto group modification go effectively to completion, under the conditions in which PKSs operate; and that in every module the downstream transfer of product to the KS of the next module is delayed because it remains loaded until the modification reactions have had time to reach completion The term ‘retardation control’ has been suggested for this proposed effect [5] The implied high level of loading in all the modules of modular PKS multienzymes is consistent with the suggested ‘leaky hose’ explanation for the release of biosynthetic intermediates from the mupirocin PKS when downstream catalytic sites are blocked [40] Maintenance of fidelity in the reductive steps relies not just on suppression of iteration, and of premature transfer of incompletely reduced polyketide chains to the next extension module, but also on precise control of reaction stereospecificity by way of molecular recognition between substrates and individual ketoreductase, dehydratase and enoylreductase domains [10,41–44] Perturbation of these interactions leads to inactivation, or to the generation of aberrant products, both in vivo [12,42] and in vitro [43] 7064 Evidence for coordination of condensation and ketoreduction In previous work [44] the operation of a single extension module from DEBS was studied in vitro by mixing individually expressed and purified domains (ACP, KR) and di-domains (KS-AT) This flexible approach allowed various combinations of each type of domain to be assayed and easily analyzed, and for individual steps to be deconvoluted For example, when a KS-AT di-domain was incubated with a diketide thioester substrate, methylmalonyl-CoA and ACP, keto triketide attached to the ACP was efficiently formed We therefore expected that when DKS was incubated with substrates in the absence of NADPH, the ACP1 would be found to carry the keto diketide However, under the conditions used, we found only the building blocks loaded on the KS and ACP1 domains respectively, and no diketide intermediate Given the surprising nature of this result, the experiment was repeated many times, always with the same outcome It appears that either the keto diketide intermediate is subject to very rapid release by the TE, or the condensation step is seriously inhibited in the absence of NADPH The former explanation can be ruled out because no evidence for a ketoacid by-product was found in any of our careful searches for by-products in early investigations of the diketide synthase in vivo or in vitro [9,45] The possible existence of an unexpected allosteric effect that inhibits the condensation step therefore deserves consideration The missing ingredient, NADPH, would be expected to bind to the KR domain, not to either of the domains involved in the condensation step Any effect is therefore remote and must depend on the quaternary structure A similar inhibition of loading of methylmalonate onto an ACP was also reported in a study of the epothilone synthase, and again the effect was attributed to the quaternary structure [33] Figure shows a schematic representation of the arrangement of domains within the extension module of the DKS, based originally on modelling and detailed proteolytic studies of DEBS that established the homodimeric nature of PKSs [46], and incorporating subsequent evidence from functional complementation [47], NMR studies [26] and X-ray crystal structures of intact animal FAS and of DEBS domains and di-domains [8,22] There is now a consensus that the two polypeptide chains in a homodimeric PKS are aligned ‘tail to tail’, as well as ‘head to head’, in each module, as predicted [46] In the absence of a crystal structure for an intact PKS module, it remains unclear how the two KS domains and the two ACP domains are able to approach FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS H Hong et al Changing patterns of covalent active-site occupancy AT1 KR1 KS1 ACP KS1 ACP AT1 TE TE KR1 closely enough to co-operate in the condensation, but one suggestion is shown in Fig The two ACP domains move, perhaps in unison, along the axis through a central passage, and so make contact with the upstream pair of KS domains The double-helical, rope-like twist of the two chains provides evidence that the ACP of one chain makes contact with the KS of the other [46,47] The cartoon shown here is equivalent to the earlier ribbon representation [46], except that the two-fold axis of symmetry runs horizontally rather than vertically, and the alternative direction of the helical twist is adopted in accordance with that established from the recent X-ray structure produced for the KS-AT di-domain [24] In this working model of the DKS, the AT and KR domains form a ‘collar’ surrounding the backwards and forwards path of travel of the two ACP domains, as they interact with their various catalytic partners The collar shelters the central region and protects the biosynthetic intermediates from the surrounding aqueous medium If the collar can expand and contract to control the lateral passage of the ACP domains, there exists a possible mechanism by which the presence of NADPH might enable the condensation step by binding to the KR and inducing a conformational change that opens the collar The AT domain may also be involved in such movements, as although the KS-AT linker exists with a tightly folded tertiary structure in the X-ray structure of the di-domain [24], this interdomain region is readily cleaved by the mild conditions of proteolysis conditions used in the present study It is tempting to speculate that the absence of methylmalonyl loading of ACP1 when a readily converted substrate (propionyl-CoA) was used, compared with the significant level of methylmalonyl loading of ACP1 with a Fig Proposed quaternary structure of the DKS chain-extension module based on the topology of the Cambridge Double Helical Model The two identical chains of the homodimeric structure are differentiated by red and blue colouring The KS domains have strong homodimeric interfaces (purple blocks) and are placed in contact at the ‘head’ of the structure The strongly homodimeric TE domains are also placed in contact with each other at the ‘tail’ The remaining three domains are not homodimeric and so can move away from the common axis running through the pairs of KS and TE domains The pair of ACP domains, however, are held close to the TE domains by short linker regions and so must remain in close proximity to each other and to the axis To aid visualization, the two domains, KR and AT, in the mid-section of the homodimer, are shown as small black blobs rather than as coloured spheres of appropriate size These domains are sited away from the axis of the proposed structure to free up a central passage The pair of ACP domains can now make contact with the pair of KS domains by moving parallel to the axis with the TE domains in tow The structure is also given a helical twist of 180 degrees in accordance with evidence that the ACP of one chain interacts with the KS of the opposite chain In the resulting quaternary structure, each ACP domain can access the appropriate KS domain for the condensation step (and other domains in succession through the chain extension cycle) by moving backwards and forwards (dashed green arrows) along the axis within the central core of the structure Because of the restricting effect of the short ACP to the TE linker, and the anticipated need for co-ordinated movements of the two looped-out domains, it is likely that the pair of ACP domains move backwards and forwards in tandem, rather than independently As a result, the successive reactions of the chain-extension cycle on the two chains might also be constrained to operate in tandem At the start of each step of the chain-extension cycle, the pair of ACP domains would be loaded with identical intermediates and both would bind with the appropriate domain for the next step The first intermediate to complete the reaction would be free to leave its catalytic domain, but its ACP would stay put until the second intermediate had completed the same operation Both intermediates are now free of the catalytic sites and the two ACP domains would then move in tandem to co-operate with the next pair of catalytic domains In the Figure, the relative positioning of the KS and AT domains conforms to that shown in the X-ray structure of an isolated KSAT fragment [24] with the AT domains in an outer position, remote from the axis However, the KS–AT linker undergoes facile proteolysis, so it must be in equilibrium with an unfolded form, not revealed in the X-ray image With the linker unfolded, the two AT domains would be free to move to inner positions closer to the axis In the outer position, they would reload with methylmalonate The subsequent move to an inner position (the original cartoon illustrating the structural principles of the Double Helical topology showed this quaternary conformation [46]) would facilitate transfer of the methylmalonate to the ACP, and, in addition, would block premature access of the unloaded ACP thiol to the KS active site, a situation that would lead to skipping of the module These predictions of co-ordinated movements of domains in the Cambridge Double Helical Model are consistent with the intriguing patterns of occupancy of ACP1 that are revealed in this investigation FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS 7065 Changing patterns of covalent active-site occupancy H Hong et al less readily converted substrate (valeryl-CoA), is preliminary evidence of a threshold level (50%) of diketide attachment to one of a pair of ACP1 domains being sufficient to suppress the movement of the unloaded partner ACP1 towards the AT1 domain for re-acylation to occur A requirement for the ACP domains to move in tandem could neatly account for such effects Unfortunately, in cutting up a homodimeric megasynthase to facilitate MS analysis, a crucial aspect of the loading information is destroyed: the analysis reveals the average level of loading across all the individual domains in the homodimeric species, but, in modules that are only partly loaded, it does not reveal how vacant and loaded sites are distributed within individual multi-enzymes There is a pressing need to develop MS protocols for analyzing larger proteins It may then be possible to study loading patterns in intact dimeric multi-enzymes, or at least in fragments that retain the homodimeric bonding that exists in the intact systems Concluding remarks Limited proteolysis followed by LC ⁄ ion-trap MS is a powerful and convenient technique for establishing features of PKS catalysis that are not readily accessible by other means The discovery that the first condensation reaction on the DEBS becomes an additional bottleneck with an unnatural starter acid provides a rational basis for efforts to improve productivity Thus, alteration of the AT domain of the loading module would be unlikely to remedy the limitation, whereas replacement of the KS by one normally operating with longer acyl chains might so Turnover on the DKS with its normal starter acyl unit is clearly regulated by the rate of release of the diketide product by the TE domain Studies of the extent of loading of multienzymes with more than one module are needed to establish if product release is indeed a general basis of regulation in PKS operations, especially in more fully evolved systems, such as the DEBS The proposal that there are two forms of control resulting from high levels of occupancy of active sites by intermediates, congestion control and retardation control, is based on the direct evidence that the KS domain is fully occupied under conditions of synthesis The evidence for the proposed quaternary effects, suppression of condensation and tandemization, is more circumstantial but deserves further study MS also has the potential to play a major supporting role in structural studies of modular PKS multienzymes It provides a method of quality control in preparing samples for structural studies by NMR, elec7066 tron microscopy, or X-ray crystallography This check on structure will be particularly desirable in the preparation of derivatized forms of multienzymes that have natural or unnatural ligands attached to the active sites of selected domains Experimental procedures Purification of the DEBS-derived DKS The expression (in Escherichia coli) and purification of the engineered DKS (comprising the first extension module of DEBS1, covalently linked to the C-terminal TE domain from DEBS 3) have been previously described [9,37] In this construct the extension module ACP (referred to here as ACP1) is a hybrid of ACP1 and ACP6, in order to preserve the native linker between the TE domain and the ACP Limited proteolysis and LC ⁄ MS analysis Limited proteolysis was performed at a protein ⁄ trypsin ratio of 80:1 (w ⁄ w) at 30 °C for After digestion, the mixture was immediately injected onto a pre-equili˚ brated C4 column (4.6 · 250 mm, 300A; Vydac, Hesperia, CA, USA) and proteins were eluted with a linear gradient of 35–55% acetonitrile (containing 0.1% trifluoroacetic acid) over 40 The analysis was performed using online LC ⁄ MS on an ion-trap instrument (LCQ Classic; ThermoFinnigan, San Jose, CA, USA) xcalibur 1.0 (ThermoFinnigan) software was used to operate the system, and bioworks 1.0 software (ThermoFinnigan) was used for mass deconvolution The detailed conditions for limited proteolysis and LC ⁄ MS analysis have been described previously [37] Substrate specificity for the chain-extender unit A mm concentration of malonyl-CoA or (RS)-methylmalonyl-CoA was incubated with lm DKS in a total volume of 30 lL, containing 400 mm potassium phosphate (pH 7.4), mm EDTA, mm dithiothreitol and 20% glycerol The reactions were carried out at 30 °C for 10 After the incubation, samples were immediately subjected to tryptic digestion and analysed using LC ⁄ MS Purification of commercial methylmalonyl-CoA Commercial methylmalonyl-CoA was dissolved in distilled deionized (MQ) water (Millipore, Billerica, MA, USA), and loaded onto a reverse-phase C18 column (Prodigy C18, 4.6 · 250 mm, l; Phenomenex, Torrance, CA, USA) Methylmalonyl-CoA and propionyl-CoA were separated by FEBS Journal 276 (2009) 7057–7069 ª 2009 The Authors Journal compilation ª 2009 FEBS H Hong et al Changing patterns of covalent active-site occupancy a linear gradient of 5–20% acetonitrile, containing 0.1% trifluoroacetic acid, over 20 Methylmalonyl-CoA was eluted about before the propionyl-CoA Fractions containing methylmalonyl-CoA were pooled, lyophilized and kept at )20 °C Purified methylmalonyl-CoA was redissolved in MQ water before use, and kept on ice adjusted to pH 6–7 with formic acid and analyzed using LC ⁄ MS The analysis was performed using the protocol described above except that after 20 min, the mass spectrometer was set up in a single full-scan mode, with scan range from m ⁄ z 600 to 2000, to monitor the ACPTE Observation of intermediates on the DKS multienzyme Acknowledgements DKS (5 lm) was incubated with mm acyl-CoA, mm methylmalonyl-CoA, and mm NADPH in a total volume of 50 lL, containing 400 mm potassium phosphate (pH 7.4), mm EDTA, mm dithiothreitol and 20% glycerol After reaction at 30 °C for 10 min, the DKS was subjected to tryptic digestion and LC ⁄ MS analysis Propionyl-CoA, n-butyryl-CoA and n-valeryl-CoA were used individually to supply starter units When n-butyryl-CoA and n-valerylCoA were used, purified methylmalonyl-CoA was applied to avoid minor contamination of propionyl-CoA in the commercial methylmalonyl-CoA Off-loading and analysis of the b-OH diketide from the diketide ACP-TE intermediate The conditions for off-loading and analysis of the b-OH diketide were optimized on a model system as follows A sample (2 nmol) of b-OH diketide-SNAC (prepared as described previously [48]) was dissolved in 400 mm potassium phosphate buffer (pH 7.4) Then, lL of hydrazine was added to a total volume of 25 lL After allowing the reaction to proceed at room temperature for h, the sample was adjusted to pH 6–7 with formic acid and the mixture was analyzed using LC ⁄ MS The analysis was performed on a C18 column (Prodigy C18, 2.0 · 250 mm, l; Phenomenex) with a gradient of 2–50% acetonitrile containing 0.1% trifluoroacetic acid, over 20 The LCQ mass spectrometer was set up in two scan modes: full scan mode scanning from m ⁄ z 50 to 200; and MS ⁄ MS mode with m ⁄ z 147.1 as the precursor ion and collision energy at 20.5% Fractions containing the hydrazide reaction product were also collected, 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Journal compilation ª 2009 FEBS 7069 ... 55 N? ?A N? ?A 0a 0a N? ?A N? ?A 58 4 9a 2 8a a Methylmalonyl-CoA Malonyl-CoA Propionyl-CoA; methylmalonyl-CoA; NADPH Butyryl-CoA; methylmalonyl-CoA; NADPH Valeryl–CoA; methylmalonyl-CoA; NADPH Propionyl-CoA;... chain-extension AT and ACP domains was considerably less than 100% As the AT-catalyzed reaction is readily reversible, the extent of loading of methylmalonate on the AT and ACP domains is probably... changes during catalysis [22,23] Both animal FAS and modular PKS are functional homodimers, which raises additional questions about the interactions between the active sites of an identical pair of