Báo cáo khoa học: Role of DptE and DptF in the lipidation reaction of daptomycin ppt

12 600 0
  • Loading ...
    Loading ...
    Loading ...

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Tài liệu liên quan

Thông tin tài liệu

Ngày đăng: 07/03/2014, 04:20

Role of DptE and DptF in the lipidation reactionof daptomycinMelanie Wittmann, Uwe Linne, Verena Pohlmann and Mohamed A. MarahielDepartment of Chemistry ⁄ Biochemistry, Philipps-University Marburg, GermanyDaptomycin is a clinically important semi-syntheticderivative of the A21978C branched cyclic lipopeptideantibiotics produced by Streptomyces roseosporus [1] .Acidic lipopeptide antibiotics present a new class oftherapeutic agents that includes compounds such ascalcium dependent antibiotic (CDA) [2], A54145 [3,4]and friulimicin [5,6] with a unique mechanism ofaction. Daptomycin binds to Gram-positive cellmembranes via its lipid moiety, followed by calcium-dependent insertion and oligomerization. Subsequently,oligomers form ion channels that disrupt the bacterialmembrane potential, leading to rapid cell death [7,8].Daptomycin comprises a 13-amino acid peptide corecoupled to a fatty acid moiety (Fig. 1). The peptidecore is assembled nonribosomally by dptA and dptBC.The thioesterase DptD of the daptomycin biosyntheticgene cluster catalyses the cyclization reaction betweenthe hydroxyl group of Thr4 and the C-terminalKyn13, resulting in a ten-membered ring [8]. More-over, several ORFs localized within the gene clusterare associated with the biosynthesis of non-proteino-genic amino acids and incorporation of the fatty acidmoiety [1].All acidic lipopeptides (except CDA) producedin vivo show some flexibility with respect to the lengthand branching of their N-terminally attached fatty acidgroups (Fig. 1). The activity of lipopeptide antibioticsas well as the toxicity towards eukaryotic cells stronglydepends on the nature of the acyl moiety [9,10]. Thefine tuning between these two features is of consider-able importance for the development of selectivepotent drugs.The biosynthesis of the peptide core of these acidiclipopeptides via nonribosomal peptide synthetases(NRPSs) is well understood, but little is known aboutthe incorporation of the acyl residue into the finalproduct [11,12]. As revealed by sequence comparison,the initiation modules of such NRPSs contain uniqueKeywordsacidic lipopeptide antibiotics; AMP ligase;daptomycin; lipidation reaction;nonribosomal peptide synthetasesCorrespondenceM. A. Marahiel, Department ofChemistry ⁄ Biochemistry, Philipps-UniversityMarburg, Hans-Meerwein-Strasse, D-35043Marburg, GermanyFax: +49 6421 2822191Tel: +49 6241 2825722E-mail: marahiel@chemie.uni-marburg.de(Received 4 June 2008, revised 29 August2008, accepted 1 September 2008)doi:10.1111/j.1742-4658.2008.06664.xDaptomycin and A21987C antibiotics are branched, cyclic, nonribosomallyassembled acidic lipodepsipeptides produced by Streptomyces roseosporus.The antibacterial activity of daptomycin against Gram-positive bacteriastrongly depends on the nature of the N-terminal fatty acid moiety. Twogenes, dptE and dptF, localized upstream of the daptomycin nonribosomalpeptide synthetase genes, are thought to be involved in the lipidation ofdaptomycin. Here we describe the cloning, heterologous expression, purifi-cation and biochemical characterization of the enzymes encoded by thesegenes. DptE was proven to preferentially activate branched mid- tolong-chain fatty acids under ATP consumption, and these fatty acids aresubsequently transferred onto DptF, the cognate acyl carrier protein. Addi-tionally, we demonstrate that lipidation of DptF by DptE in trans is basedon specific protein–protein interactions, as DptF is favored over other acylcarrier proteins. Study of DptE and DptF may provide useful insights intothe lipidation mechanism, and these enzymes may be used to generatenovel daptomycin derivatives with altered fatty acids.AbbreviationsCDA, calcium dependent antibiotic; PKS, polyketide synthase.FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS 5343condensation (CIII) domains that are thought to cata-lyse N-acylation of the first amino acid in the peptidechain [13]. However, the fatty acid moiety must beactivated prior to being incorporated into theproduct. Two classes of enzymes are known tocatalyse such reactions. One class, acyl CoASHsynthetases, recognize and activate fatty acids as acyladenylates (acyl AMPs), and subsequently couplethem to coenzyme A (CoASH). The second class,fatty acyl ACP ligases, activate and transfer fattyacids from acyl AMP to cognate acyl carrier proteins(ACPs) [14,15].The genes dptE and dptF are localized immediatelyupstream of the NRPSs of A21987C. The resultingproteins DptE and DptF were predicted to beinvolved in the lipidation reaction based on sequencesimilarity [1]. DptE is similar to other adenylate-forming enzymes such as acyl CoASH synthetases,and DptF is a putative ACP. Both proteins arethought to be important for the initiation of dapto-mycin biosynthesis [1,16].In this study, we describe the biochemical character-ization of DptE as an acyl ACP ligase, and demon-strate transfer of various fatty acids onto the ACPencoded by dptF (Fig. 2). This biochemical character-ization of the lipidation mechanism during acidiclipopeptide biosynthesis may facilitate engineering ofnew derivatives with altered activities.ResultsInitial biochemical characterization of DptE andDptFDptE shares approximately 20% sequence identity withseveral members of the acyl AMP ⁄ CoASH ligase super-family [17]. These enzymes catalyse the formation of fattyacyl AMP ⁄ CoASH from a fatty acid substrate, ATP andCoASH in a Mg2+-dependent two-step reaction [17–19].In general, a fatty acyl adenylate intermediate is formedin the first step, followed by conversion of the fatty acyladenylate to fatty acyl CoA with release of AMP.Fig. 1. Chemical structures of the lipopeptide antibiotics daptomycin, A54145 and CDA, and their natural fatty acid moieties. Daptomycinand A54145 are naturally produced with various fatty acid side chains. For daptomycin, the major fatty acids are shown. CDA is producedwith the epoxidized hexanoyl moiety exclusively.Lipidation of daptomycin M. Wittmann et al.5344 FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBSDptE was cloned into the pBAD102 ⁄ D-TOPOÒvector and overexpressed in Escherichia coliBL21(DE3). The C-terminally His6-tagged and N-ter-minally thioredoxin-fused protein was purified, yielding4.4 mgÆL)1of culture. The identity of the protein wasconfirmed by SDS–PAGE (Fig. 3) and mass spectro-metry (Table 1). An initial fatty acid-dependentATP ⁄ PPiexchange assay according to functionallyrelated adenylation domains of NRPSs showed noactivity (data not shown). To determine whetherCoASH is the physiological substrate of DptE andrequired for enzyme activity, we determined theactivity of DptE with ATP, MgCl2and CoASH undervarious conditions. However, no acyl CoA was detect-able by HPLC-MS (data not shown).As we were not able to detect any in vitro activity ofDptE using ATP ⁄ PPiexchange assays, and no lipidationof CoASH was observed in the presence of fatty acids,we next focused on the transcriptionally coupled dptF,which encodes a stand alone putative ACP [20]. ACPscontain the modestly conserved motif GxDS(I ⁄ L), inwhich the serine residue is post-translationally modifiedby covalent attachment of a 4¢-phosphopantetheingroup [21,22]. The motif present in DptF is GLDSV,indicating that this putative ACP domain is one of thefew ACPs in which valine replaces isoleucine (I) orleucine (L) in the conserved sequence. To determinewhether DptF is the putative partner of DptE, weexpressed dptF using the pQTev vector in E. coli andpurified the resulting ACP as an N-terminal His7 fusionprotein (Fig. 3) with a yield of 9.5 mgÆL)1of culture.The identity of the protein was proven by SDS–PAGE+ decanoic acid DptE +ATP PPi O O– 7 SH holo-ACPDptF DptA DptBC DptD daptomycin DptF S O decanoyl-S-ACP7 Mg2+ AMP DptE O O 7 AMP Fig. 2. Proposed mechanism for thelipidation of daptomycin by DptE and DptF.Decanoic acid is activated by the putativeadenylating enzyme DptE under ATP con-sumption. The fatty acid is then transferredonto the acyl carrier protein DptF. The Cdomain of DptA is predicted to catalyse thecondensation reaction between the fattyacid and tryptophan.kDaDptEaDptFhDptFaLipDkDahLipDhAcpK8025201530405060100150Fig. 3. Coomassie blue-stained SDS–PAGE gel of purified apo-DptF(aDptF, 13.5 kDa), holo-DptF (hDptF, 13.8 kDa), apo-LipD (aLipD,11.1 kDa), holo-LipD (11.5 kDa), hAcpK (B. subtilis, holo form;10.9 kDa) and DptE (80.5 kDa). SDS–PAGE was performed using aNuPAGE 4-12% Bis-Tris gel (Invitrogen). The protein ladder wasfrom New England Biolabs (P7703, 10-250 kDa).Table 1. [M+H]+mass values for the proteins, substrates andproducts.[M+H]+(Da)Sample Mass observed Mass calculatedapo-DptF 13 491.6 13 491.9holo-DptF 13 831.7 13 831.9apo-LipD 11 140.2 11 140.4holo-LipD 11 480.4 11 480.5apo-AcpK 10 561.5 10 561.0holo-AcpK 10 901.6 10 901.0decanoyl-DptF 13 986.9 13 986.9decanoyl-LipD 11 635.6 11 635.7decanoyl-AcpK 11 056.7 11 056.8M. Wittmann et al. Lipidation of daptomycinFEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS 5345and tryptic digestion followed by mass spectrometry.Subsequently, DptF was incubated with the promiscu-ous 4¢-phosphopantetheinyl transferase Sfp from Bacil-lus subtilis and fluoresceinyl CoA [23]. The successful4¢-phosphopantetheinylation of DptF was monitored bythe in-gel fluorescence of the reaction mixture (Fig. 4).For subsequent acylation studies, holo-DptF wasproduced in the sfp-containing E. coli strain HM0079[24]. The in vivo modification of DptF by Sfp resultedin 100% conversion of apo-DptF to holo-DptF asestimated by tandem fourier transform ion cyclotronresonance-MS (Fig. 5 and Table 1).Lipidation of DptF by DptEInitially, 50 lm holo-DptF was incubated with 500 lmdecanoic acid, 10 mm MgCl2,1mm ATP and 1 lmDptE (Fig. 5). The reaction mixture was quenched with10% formic acid after 10 min and subjected to HPLC-ESI-MS analysis (Table 1). DptF was quantitativelyacylated with decanoic acid. Subsequently, we deter-mined the pH and temperature for maximum forma-tion of decanoyl-S-ACP catalysed by DptE. Suitablereaction conditions were determined to be pH 7.0 and37 °C, in agreement with those reported for other acylAMP ⁄ ACP ⁄ CoASH ligases [15,25]. Omitting DptE orATP abolished the acylation of DptF completely.These results indicate that decanoic acid is activated asa fatty acyl AMP and subsequently transferred ontoholo-DptF by the acyl ACP synthetase DptE. To detectthe adenylate intermediate, we repeated the reactionwith apo-DptF rather than holo-DptF, which shouldlead to accumulation of the acyl adenylate intermedi-ate. The reaction was stopped using 10% formic acidand subjected to LC-MS to detect decanoic AMP (datanot shown). However, we were not able to detect theadenylate intermediate using this approach. Next, weperformed an ATP ⁄ PPiexchange assay with apo-DptFin the presence of phosphate buffer. Control reactionswere performed without radioactively labelled PPi,DptE, apo-DptF, MgCl2, or ATP. In the presence ofapo-DptF, we observed an approximately 100-foldhigher activity of DptE compared to the control reac-tions (Fig. 6). The above-mentioned conditions wereused for determination of steady-state kinetic para-meters. The KMand kcatvalues of DptE for holo-DptF(with concentrations between 2.5 and 250 lm) were29.4 lm and 7.4 min)1under decanoic acid satura-tion (500 lm), resulting in a catalytic efficiency of0.25 min)1Ælm)1. Addition of CoASH to the reaction10152030Sfp++––50kDakDaapo-DptFapo-AcpKSDS-PAGE UV-irradiation at 312 nmkDakDaapo-DptFapo-AcpK+– + –Fig. 4. In vitro phosphopantetheinylation of apo-DptF and apo-AcpK. Coomassie blue-stained SDS–PAGE gel (left) and in-gel fluo-rescence (right) of the fluoresceinyl-ACP. (+) indicates the reactionwith Sfp; ()) indicates the reaction without Sfp.m/z13491.6apo-DptF+ Na+ KaRelative abundance13 200 13 300 13 400 13 500 13 600 13 700 13 800 13 90013 600 13 700 13 800 13 900 14 00014 10013 500Relative abundance13831.7holo-DptF+ Na+ Kam/z13986.9decanoyl-DptF+ Ka+ NaRelative abundance13 500 13 600 13 700 13 800 13 900 14 000 14 100Sfp/ CoASH/Mg2+-5'-3'-ADPDptE-AMP + PPi+ATPm/zFig. 5. Fourier transform MS spectra of apo-DptF (left), holo-DptF (middle) and decanoic acid-loaded DptF (right).0 5000 10 000 15 000 20 000 25 000 c.p.m.Assay -PPi* -DptE -ATP -MgCl2 30 000 Fig. 6. ATP ⁄ PPiexchange assay of DptE in the presence of apo-DptF, and control reactions without radioactive labeled PPi(PPi*),DptE, ATP or MgCl2.Lipidation of daptomycin M. Wittmann et al.5346 FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBSmixture did not affect the product formation activity(data not shown). Therefore, the results clearly demon-strate that holo-DptF is the cognate acceptor substrateof DptE (Table 2).Fatty acid specificity of the AMP ligase DptEHaving proven a functional interaction of DptE andDptF utilizing decanoic acid as a standard substrate,we addressed the important question of DptE specific-ity. We systematically utilized a range of linear andbranched chain fatty acids as well as hydroxy-fattyacids with various chain lengths and varied the concen-trations between 2.5 and 500 lm. Kinetic constantswere determined by Michaelis–Menten fitting of thedata sets. The summarized kinetic data, which wereobtained under ATP and holo-DptF saturation, arepresented in Table 2. As expected, the fatty acids(branched and linear, between 10 and 12 carbon units)that are known to be present in naturally producedA21987C lipopeptides and in the drug CubicinÒ (dap-tomycin formulated for injection) were observed to beexcellent substrates, with KMvalues ranging from 8 to20 lm and kcatvalues between 3.4 and 18.3 min)1.Catalytic efficiencies were 0.29–0.95 min)1Ælm)1. Thesevalues are in good agreement with those observed forother systems in which a fatty acyl ACP synthetaselipidates a cognate holo-ACP in trans [26]. Octanoicacid, tetradecanoic acid and the 3-hydroxy fatty acid,which have not been reported as occurring in thenatural compound, were relatively poor substrates,with KMvalues 2–13-fold higher than those for fattyacids naturally found in A21987C. Hexanoic acid,palmitic acid and 15-methylhexadecanoic acid were notaccepted by DptE.In summary, DptE is capable of transferring avariety of fatty acids to the cognate ACP DptF in vitro.The kinetic data presented in this study indicate thatDptE has a general preference for linear fatty acidswith chain lengths between 8 and 14 carbon units,particularly iso ⁄ anteiso-branched chain fatty acids andTable 2. Kinetic parameters for steady-state analysis of the DptE-catalysed lipidation of DptF determined at varying concentrations offatty acids or DptF, LipD and AcpK.Substrate KM(lM) kcat(min)1) kcat⁄ KM(min)1ÆlM)1)LinearaC8 65.0 ± 1.2 7.2 ± 0.5 0.11 ± 0.01C10 8.2 ± 0.4 3.4 ± 0.2 0.42 ± 0.04C12 10.9 ± 0.3 3.1 ± 0.1 0.29 ± 0.01C14 26.6 ± 0.9 1.3 ± 0.2 0.05 ± 0.01Branchedbiso-C10 19.3 ± 0.5 17.9 ± 0.5 0.93 ± 0.05iso-C12 19.2 ± 0.4 18.3 ± 0.5 0 .95 ± 0.05iso-C13 16.1 ± 0.6 15.3 ± 0.7 0.95 ± 0.08anteiso-C12 14.1 ± 0.8 13.1 ± 0.2 0.93 ± 0.08Hydroxylated3OH-C12 114.2 ± 4.2 5.1 ± 0.4 0.04 ± 0.01ACPsholo-DptF 29.4 ± 0.4 7.4 ± 0.2 0.25 ± 0.01holo-LipD 135.0 ± 0.5 6.3 ± 0.3 0.05 ± 0.03holo-AcpK ND ND NDaSubstrates C6 and C16 were not activated.bSubstrate anteiso-C16 was not activated.+ Na 10901.6 holo-AcpK 10 600 10 500 10 700 10 800 10 900 11 000 Relative abundance m/z 10901.6 holo-AcpK + Na 10561.5 apo-AcpK m/z 10 600 10 700 10 800 10 900 11 000 11 100 11 200 11 300 Relative abundance -5'-3'-ADP Sfp/ CoASH/Mg2+Fig. 7. AcpK expressed in its active holo form in M15 ⁄ pRep4-gspcells (HM404). Only approximately 40% of AcpK is expressed inthe holo form (upper). Phosphopantetheinylation of apo-AcpK withSfp after expressing in M15 ⁄ pRep4-gsp cells (HM404) (lower).M. Wittmann et al. Lipidation of daptomycinFEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS 5347decanoic acid, while long chain fatty acids such aspalmitic acid or 15-methylhexadecanoic acid are notrecognized at all. Hydroxylated fatty acids areaccepted, but with lower efficiencies.ACP specificity of the acyl ACP ligase DptEThe results presented above confirm that DptE acti-vates various fatty acids and transfers them ontoDptF. To address the question of the specificity ofDptE towards ACPs, we utilized as alternative ACPsLipD, an ACP that is involved in friulimicin biosyn-thesis and shows approximately 31% sequence identitywith DptF, and holo-AcpK (Fig. 7) from B. subtilis,which shares approximately 13% sequence identitywith DptF. Mass spectrometry analysis of the assayedholo-AcpK showed no product formation. In areaction mixture containing both DptF and AcpK,acylation of DptF exclusively was observed (data notshown). LipD was only partially acylated in presenceor absence of DptF. For better comparison ofthe reaction velocities obtained with DptF and LipD,we performed kinetic studies. To determine kineticdata for LipD, this protein was expressed in vivo in itsactive holo form (see Experimental procedures). Thereaction mixtures contained 1 mm ATP, 10 mmMgCl2, 2–250 lm holo-LipD, 1% dimethylsulfoxideand 500 lm decanoic acid. Michaelis–Menten fitting ofthe experimental data set resulted in a KMof 135 lmand a kcatof 6.3 min)1. The catalytic efficiency of thetransfer reaction to LipD (0.047 min)1Ælm)1) wasapproximately five times lower than that for DptF(0.25 min)1Ælm)1) (Table 2). In conclusion, theseresults suggest that there is specific recognitionbetween DptE and DptF.DiscussionDaptomycin is a prominent member of the pharmaco-logically important class of antimicrobial acidic lipo-peptides. It has been commercialized as CubicinÒ(Cubist Pharmaceuticals Inc., Lexington, PA, USA)for the treatment of serious infections caused byGram-positive bacteria [27]. Recently, it has beenshown that the activity of these acidic lipopeptides issignificantly influenced by the length and structure oftheir fatty acid moieties [9,10]. In the fermentation ofthese natural products, some flexibility with respect tothe length and branching of the lipid side chain hasbeen observed [10]. Complete biochemical characteri-zation of the lipidation reaction may allow theengineering of lipopeptides with modified fatty acidmoieties, which could lead to new antibiotics activeagainst a wide range of bacteria, preventing damage toeukaryotic cells. For incorporation of the fatty acidmoiety into nonribosomal peptides, condensation ofthe fatty acid with the N-terminal tryptophan of thenonribosomally synthesized peptide is necessary.Here, we report the results of a steady-state kineticanalysis of DptE. The aim of this kinetic study was todetermine the specificity of DptE for various fattyacids and noncognate ACPs. The Michaelis–Mentenkinetic values indicate catalysis of the two-stepreaction with one substrate (fatty acid or ACP) undersaturating or non-saturating conditions. The kineticdata for the various fatty acids transferred onto DptFby DptE reported here indicate the preference of DptEfor those found in the naturally produced daptomycinderivatives. Additionally, it was observed that long-chain (16 carbon units or more) and short-chain fattyacids (six carbon units or fewer) are not accepted byDptE. The observation that DptE is able to activateand transfer a broad range of fatty acids fits well withresults for other fatty acid CoASH synthetases such asFaa1p from Saccaromyces cerevisiae [28] or CpPKS1-AL from Cryptosporidium parvum [26]. Faa1p func-tions in the vectorial acylation of exogenous long-chainfatty acids, and has a preference for fatty acid sub-strates with 10–18 carbons. The KMvalue of Faa1pfor oleate is 71.1 lm. The CpPKS1-AL domain hasbeen proposed to be involved in the biosynthesis of ayet undetermined polyketide. This domain also showsbroad substrate acceptance but with a preference forlong-chain fatty acids, particularly arachidic acid. Theactual substrates for the fatty acid CoASH ⁄ ACPsynthetases will be limited by the availability of fattyacids in the host organism.Interestingly, comparison of the kcat⁄ KMvalues forDptE revealed that it is five times more active with thephysiologically relevant ACP DptF than with to LipD(Table 2), and is inactive with AcpK. Therefore, thein trans lipidation of DptF appears to be the result ofspecific protein–protein communication [29].Faa1p, which functions by a common ‘ping pongBI-BI’ mechanism [30–32], showed a KMof 18.3 lmfor its cognate ACP. In the case of CpPKS1-AL, theKMfor the lipidation of ACP was 3.53 lm. Thesefindings are in good agreement with those for DptE,which has a KMof 29.4 lm for its cognate ACP.In microorganisms, various strategies exist for theactivation of fatty acids. Gokhale et al. [14,33,34]found several enzymes for fatty acid activation inMycobacterium tuberculosis. These putative enzymeswere cloned and expressed in E. coli, and two distinctclasses were found, namely fatty acyl AMP ligases andfatty acyl CoASH ligases. The AMP ligases activateLipidation of daptomycin M. Wittmann et al.5348 FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBSmetabolic fatty acids as acyl adenylates, which are sub-sequently transferred to a cognate holo-ACP domain.In contrast, the acyl CoASH ligases catalyse transferonto CoASH, forming an acyl thioester, whichsubsequently undergoes transthiolation with theHS-phosphopantetheine group of an ACP [14,33,34].The loading module of the polyketide synthase (PKS) ⁄NRPS hybrid mycosubtilin was recently characterized[35,36]. It was shown that priming of ACP1with afatty acid occurs via an acyl AMP ligase domain in cis.The latter type of fatty acid activation and loadingwas exclusively reported for PKS systems [14].Recently, lipidation of the acidic lipopeptide CDAwas investigated in vivo and in vitro [16]. However,CDA is an exception within the acidic lipopeptides, asonly 2,3-epoxy-hexanoic acid is incorporated into thefinal product, and two specific enzymes encoded byfabH3 and fabH4 are thought to synthesize hexanoicacid directly on an ACP. Two additional proteinsencoded in the CDA fab operon, HxcO and HcmO,are responsible for the subsequent epoxidation of hexa-noyl S-ACP [37].Interestingly, in the case of the lipopeptide surfactin,neither an acyl CoASH ligase-like domain nor an ACPcould be identified within the biosynthetic gene clusterusing bioinformatic tools [38]. Previously, an unknown40 kDa protein was thought to be the candidate forlipidation. However, it has been suggested that theactivated 3-hydroxymyristoyl CoA substrate is bio-synthesized by the primary metabolism. Recently, itwas reported that the acyl CoA substrate is transferredto the initiation module SrfA-A1. This transfer isstimulated by the surfactin thioesterase II SrfD [38].However, the reaction also took place in the absenceof the thioesterase, but with reduced turnover. Todate, no additional enzyme such as an acyltransferaseor an acyl CoASH ligase has been reported to beinvolved in the surfactin initiation process.Another possibility for lipidation of secondarymetabolites could be the interaction of fatty acidsynthase-like enzymes or substrates from the primarymetabolism with NRPSs or PKSs, as shown for afla-toxin produced by the fungi Asparagillus parasiticusand A. flavus [39,40]. In this example, the fatty acidsynthase-like enzymes HexA and HexB synthesizehexanoic acid from acetyl CoA and two units ofmalonyl CoA. This hexanoic acid serves as a precursorfor initiation of the PKS of aflatoxin biosynthesis.As shown here, the acyl ACP ligase DptE of thedaptomycin biosynthetic gene cluster appears todirectly select and activate cytosolic fatty acids fromprimary metabolism as fatty acyl adenylates in a mech-anism analogous to the adenylation domains ofNRPSs [41]. Subsequently, the fatty acids are trans-ferred in trans onto holo-DptF to generate fatty acylS-ACP. No lipidation was observed without ATP,confirming our conclusion that the fatty acid has to beactivated as an adenylate prior to esterfication by thecognate ACP.Interestingly, we detected a 100-fold higher activityover background in the ATP ⁄ PPiexchange assay withDptE when it was performed in the presence of nonre-active apo-DptF (approximately 26 500 c.p.m.). In theabsence of DptE we found only a marginal activity(approximately 250 c.p.m.). This leads to the conclu-sion that DptE requires DptF for its activity. We sug-gest that the reason that a fatty acid adenylateintermediate was not detected using apo-DptF in anLC-MS approach is that the back reaction was toofast or the amount of product was below the detectionlimit.Summarizing, the present study focuses on the bio-chemical characterization of DptE and DptF. Todate, we cannot rule out the possibility that similarfatty acid CoA derivatives will also be recognized bythe C domain of the initiation module of dapto-mycin. That DptE and DptF are involved in thelipidation process of daptomycin was first shown byMiao et al. [1]. In their work, the daptomycin genecluster was heterologously expressed in Streptomyceslividans. Only authentic daptomycin derivatives werefound and no derivatives with common fatty acidsof the S. lividans organism. Studies utilizing deletionmutants or biochemical studies involving the initia-tion module of daptomycin synthetase are requiredto prove whether DptE and DptF are essential forlipidation or whether there are additionally alterna-tive pathways.In conclusion, DptE was observed to recognize avariety of fatty acid moieties. After activation of thefatty acids under ATP consumption, most likely asfatty acyl AMPs, DptE subsequently catalyses specifictransfer onto the 4¢-phosphopantethein group of DptF.The observed substrate tolerance for loading a varietyof fatty acids onto the ACP will facilitate future pro-jects on the manipulation and combinatorial biosyn-thesis of acidic lipopeptides. Hopefully, the recognitionand efficient transfer of new building blocks can beachieved using DptE and DptF. This is important, asthe fatty acid moiety has been proven to have a highimpact on the bioactivity and bioselectivity of theseantibiotics [9,10]. It remains to be clarified whether allof the fatty acids activated by DptE can be incorpo-rated into the final product or whether there is aninterfering specificity of the CIIIdomain of the initia-tion module.M. Wittmann et al. Lipidation of daptomycinFEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS 5349Experimental proceduresMaterialsElectrocompetent Top10 and BL21 (DE3) E. coli cells werepurchased from Invitrogen (Carlsbad, CA, USA). Allrestriction endonucleases and T4 DNA ligase were obtainedfrom New England Biolabs (NEB GmbH, Hilden,Germany). Oligonucleotides were purchased from Operon(Operon Biotechnologies GmbH, Cologne, Germany).Plasmid DNA isolation was performed using a Qiagen spinminiprep kit (Qiagen GmbH, Hilden, Germany). DNAsequencing was performed at GATC Biotech AG(Konstanz, Germany). The plasmid pBAD102 ⁄ D-TOPOÒwas purchased from Invitrogen. The pQTev vector, whichis a derivative of pQE60, was purchased from Qiagen.Fatty acids were purchased from Larodan (LARODANFine Chemicals AB, Malmoe, Sweden). All other materialswere purchased from Sigma-Aldrich (Sigma Aldrich ChemieGmbH, Munich, Germany).DNA isolationS. roseosporus NRLL 11379 was inoculated in nutrient brothand grown at 37 °C for 48 h with agitation. Genomic DNAwas isolated using a DNeasy Blood and Tissue kit (Qiagen).Cloning and expression of DptFThe 270 bp dptF gene was amplified by PCR from S. roseosp-orus NRLL 11379 genomic DNA using high-fidelity PhusionDNA polymerase (Finnzymes, Espoo, Finland) and primersdptF-for (5¢-TATGGATCCAACCCGCCCGAAGC GGTC-3¢)and dptF-rev (5¢-ATAGCGGCCGCGGTGCGGTCGGCCAACTG-3¢) (underlining indicates artificial BamHI and NotIrestriction sites). The amplified product was purified on a1.2% agarose gel using a PCR gel extraction kit (Qiagen),digested with BamHI and NotI, and ligated into the samesites of the pQTev vector to yield the plasmid pQTev-dptF.The integrity of the plasmid was confirmed by sequencing.The resulting plasmid was used to transform E. coli BL21(DE3) or E. coli HM0079 [24] for gene expression. The cul-tures were grown in LB medium supplemented with100 lgÆmL)1ampicillin. Cultures were grown at 37 °Ctoanattenuance at 600 nm of 0.5, and then the temperature wasdecreased to 30 °C and gene expression was induced by addi-tion of 0.1 mm isopropyl thio-b-d-galactoside (IPTG, finalconcentration). Cultures were grown for an additional 4 hand then harvested by centrifugation (4000 g,4°C, 15 min).Cloning and expression of DptEThe 1795 bp dptE gene was amplified from Strepto-myces roseosporus NRLL 11379 genomic DNA using high-fidelity Phusion DNA polymerase (Finnzymes) and primersdptE-for (5¢-CACCATGAGTGAGAGCCGCTGTGCCGG-3¢; underlining indicates the sequence overhang for theTOPO cloning) and dptE-rev (5¢-CGCGGGGTGCGGATGTGGAG-3¢). The amplified product was purified from a0.8% agarose gel using a PCR gel extraction kit (Qiagen),and ligated into pBAD102 ⁄ D-TOPOÒ (Invitrogen) accord-ing to manufacturer’s instructions to yield the plasmidpBAD102 ⁄ D-TOPO-dptE. The integrity of the plasmid wasconfirmed by sequencing. The resulting plasmid was usedto transform E. coli BL21 (DE3) for gene expression. Thecultures were grown at 37 °C in LB medium supplementedwith 100 lgÆmL)1ampicillin to an attenuance at 600 nm of0.5. The temperature was then decreased to 28 °C, and geneexpression was induced by addition of 0.1 m m IPTG (finalconcentration). Cultures were grown for an additional 4 hand then harvested by centrifugation (4000 g,4°C, 15 min).Purification of recombinant expressed proteinsDptE and DptFFor purification of DptE and DptF, cell pellets from 1 litreof culture were resuspended in 10 mL of buffer A (50 mmphosphate buffer, 300 mm NaCl, pH 7.0) and disruptedusing a French press (SLM Aminco; Thermo FrenchÒpress,G. Heinemann Labortechnik, Schwaebisch Gemuend, Ger-many). Insoluble cell debris was removed by centrifugation(17 000 g,4°C, 45 min). Purification of the His-taggedfusion proteins using Ni2+-NTA superflow resin (Qiagen)was performed on an FPLC system (Amersham PharmaciaBiotechnology, Amersham, UK) according to manufac-turer’s standard protocol. Briefly, fractions containing therecombinant proteins were monitored by SDS–PAGE,pooled, and dialysed against phosphate buffer with 100 mmNaCl using HiTrapÔ desalting columns (GE Healthcare Eur-ope GmbH, Freiburg, Germany). The recombinant proteinswere then concentrated using membrane-based AmiconÒUltra-15 concentrators (Millipore GmbH, Schwalbach,Germany) with a molecular mass cut-off of 10 kDa (DptF)and 50 kDa (DptE). Protein concentrations were determinedby NanoDropÒ spectrophotometer ND-1000 (PeqLabBiotechnologie GmbH, Erlangen, Germany) measurements.The affinity-purified proteins were stored at )80 °C.In vitro 4¢-phosphopantetheinylation of apo-DptFA reaction mixture containing 200 lm fluoresceinyl CoA orCoASH [42], 50 lm DptF, 10 mm MgCl2and 0.5 lm recom-binant Bacillus subtilis 4¢-phosphopantetheine transferaseSfp in assay buffer (50 mm phosphate buffer, 100 mmNaCl, pH 7.0) was incubated at 37 °C for 5–30 min andanalysed on an SDS–PAGE gel by measuring the in-gelfluorescence. The Sfp substrate fluoresceinyl CoAwas generated as previously described [23]. The CoASHLipidation of daptomycin M. Wittmann et al.5350 FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBSmodification of DptF was verified by ESI-MS usingan LTQ-FT mass spectrometer (Thermo Fisher Scientific,Bremen, Germany).ATP-pyrophosphate exchange assayThe ATP ⁄ PPiexchange reaction was used to determine theactivity and substrate specificity of DptE. For all assays,the enzyme concentration varied from 300 nm to 1 mm,and the ATP concentration was at a saturating level of2mm. All reactions were performed at 37 °C. ATP ⁄ PPireactions were performed for 30 s to 1 min. Reactionmixtures contained 50 mm Hepes, pH 8.0, 100 mm NaCl,10 mm MgCl2, 500 nm decanoic acid and 300 nm to 1 lmDptE (in a final volume of 100 lL). The reaction wasinitiated by addition of ATP, 50 lm tetrasodium pyrophos-phate (NaPPi) and 0.15 lCi (16 CiÆmmol)1) of tetrasodiumpyrophosphate (radioactive labeled PPi, Perkin Elmer,Waltham, MA, USA). The reactions were quenched byadding 500 lL of a stop mix containing 1.2% w ⁄ v acti-vated charcoal, 0.1 m tetrasodium pyrophosphate and0.35 m perchloric acid. Subsequently, the charcoal waspelleted by centrifugation (4000 g,4°C, 3 min), washedtwice with 1 mL water (vortexed for 30 s), and once with0.5 mL water. After addition of 0.5 mL water and 3.5 mLof liquid scintillation fluid (Rotiscint Eco Plus, CarlRothGmbH and Co. KG, Karlsruhe, Germany), the charcoal-bound radioactivity was determined by liquid scintillationcounting using a 1900CA Tri-carb liquid scintillationanalyser (Packard Instruments, Meriden, CT, USA).Activity assay of DptE with CoASHAcyl CoASH synthetases ⁄ ligases are thought to catalyse thethioesterification of a fatty acid with CoASH. In this study,we showed that DptE was not able to react with CoASHas a substrate. However, a typical reaction mixture(100 lL) was composed of 50 mm phosphate buffer,100 mm NaCl, 10 mm MgCl2,1mm ATP, 300 lm CoASH,500 lm decanoic acid, 1% dimethylsulfoxide and 1 mmDptE. After incubation at 37 °C for 30 min, reactions werestopped with 10 lL formic acid. The product formationwas measured by HPLC-MS. Separation of the reactionproducts was achieved on a 250 ⁄ 3 Nucleosil C8 column(3 lm, Macherey-Nagel GmbH & Co. KG, Du¨ren,Germany) by applying the following gradient at a flow rateof 0.3 mLÆmin)1[buffer A: 2mm triethylamine ⁄ water; bufferB: 2mm triethylamino ⁄ 80% acetonitrile ⁄ 20% water (v ⁄ v)],column temperature 30 °C: loading 5% buffer B, after5 min linear gradient up to 95% buffer B in 37 min, andthen holding 100% buffer B for 5 min. The product wasidentified by UV detection at 215 nm and by on-lineESI-MS analysis with an Agilent 1100 MSD (AgilentTechnologies Deutschland GmbH, Boeblingen, Germany)in the negative single ion monitoring (SIM) mode.DptE-mediated transfer of long-chain fatty acidsto holo-DptFFor this reaction, we used holo-DptF that was heterolo-gously expressed in sfp-containing E. coli HM0079 cells. Atypical reaction mixture contained 50 lm holo-DptF,250 lm fatty acid, 10 mm MgCl2,1mm ATP, 1% dimethyl-sulfoxide and 1 lm DptE in a total volume of 25 lL50 mm phosphate buffer (pH 7.0) with 100 mm NaCl. Afterincubation at 37 °C for 0–30 min, the reaction wasquenched by addition of 7.5 lL formic acid and directlyanalysed by mass spectrometry using an LTQ-FT instru-ment (Thermo Fisher Scientific), with desalting using an8 ⁄ 2 Nucleodur C4 pre-column (Macherey & Nagel). Thesolvents used were water with 0.05% formic acid andacetonitrile with 0.045% formic acid. The followinggradient was applied at a flow rate of 0.2 mL Æ min)1and acolumn temperature of 40 °C. The sample was loaded with95% buffer A for 3 min, followed by a linear gradientdown to 60% buffer A in 15 min, followed by a lineargradient down to 5% buffer A in 2 min. 5% buffer A washeld for an additional 2 min and followed by a lineargradient up to 95% buffer A in 6 min.Determination of the acyl adenylate intermediateLC-MS approachTo identify decanoic AMP by LC-MS, reactions (100 lL)containing decanoic acid (500 lm), ATP (1 lm), MgCl2(10 mm), apo-DptF (50 lm), 1% dimethylsulfoxide andphosphate buffer (pH 7.0, 50 mm) were performed at37 °C. Reactions were initiated by addition of DptE (5 lm)and stopped after 1 h by addition of 30 lL formic acid.Samples were analysed by HPLC-MS as described above.ATP/PPi-exchange approachFor activity measurements, DptE (1 lm) was rapidily mixedwith 0.15 lCi (16 CiÆmmol)1) hot PPiin the presence of500 lm decanoic acid, 1% dimethylsulfoxide, 10 mmMgCl2,1mm ATP, 10 lm apo-DptF and 5 mm NaPPiphosphate buffer (pH 7.0, 50 mm)at37°C. The enzymeactivity was also checked in the absence apo-DptF, hot PPi,DptE or MgCl2as control reactions. The reactions werestopped after 60 min by addition of 500 lL stop mix. Sam-ples were washed and analysed as described above.Determination of the kinetic parameters of DptFlipidation by DptETo determine the kinetic parameters for holo-DtpF lipida-tion by DptE, we performed the reactions under ACP satu-ration and varied the fatty acid concentrations. Dependingon the substrate, we varied the reaction time between 30 sM. Wittmann et al. Lipidation of daptomycinFEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS 5351and 4 min. The reactions were carried out at 37 °Cinatotal volume of 25 lL. Unless otherwise indicated, thereaction mixtures contained 50 mm phosphate buffer,100 mm NaCl, pH 7.0, 10 mm MgCl2,1mm ATP, 1 mmDptE, 50 lm holo-DptF, 1% dimethylsulfoxide and variousconcentrations of fatty acids (10–250 lm). The reactionswere stopped by the addition of 7.5 lL acetic acid. Theconversion rate of holo-DptF into fatty acyl S-DptF wasanalysed by LC-ESI-MS as described above. The steady-state parameters kcatand kcat⁄ KMand their standard errorswere determined using nonlinear regression with sigmaplot8.0 (Systat Software GmbH, Erkrath, Germany) to fit thedata to the Michaelis–Menten equation.Determination of DptE specificity towards otherACPsE. coli HM404 cells (E. coli M15 ⁄ pREP4-gsp transformedwith pQE60-acpK) [43] were a gift from H. D. Mootz(Fachbereich Chemische Biologie, Technische Universita¨tDortmund, Germany). The expression The E. coli HM404cells were grown in the presence of 25 lgÆmL)1kanamy-cin, induced, harvested and disrupted, and the crude cellextract was centrifuged as described above for DptF. Pro-tein purification was performed as described previously[43]. The yield of purified protein was 5.5 mgÆL)1ofculture (Fig. 7).The lipD gene was amplified from genomic DNA usingPhusion DNA polymerase (Finnzymes) and the syntheticoligonucleotide primers 5¢-AAAAAAGAATTCATGTCAGACCTCAGCACCGC-3¢ and 5¢-AAAAAAAGCTTTCAGGCGGAACGCAGCTC-3¢ (EcoRI and HindIII restric-tion sites are underlined). The resulting 291 bp PCR frag-ment was purified, digested with EcoRI and HindIII, andligated into a pET28a(+) derivative (Novagen, MerckKGaA, Darmstadt, Germany), digested with the sameenzymes. The identity of the resulting plasmid pCB28a(+)-lipD with an N-terminal hexahistidine tag was confirmed byDNA sequencing.The plasmid was used to transform E. coli strainBL21(DE3) (Novagen) and the enzyme was overproducedin LB medium supplemented with kanamycin (50 lgÆmL)1).The cultures were grown to an absorbance of 0.3 at 30 °C.The cultures were cooled to 18 °C and protein productionwas induced by the addition of IPTG to a final concentra-tion of 0.1 mm. The cultures were incubated for a further18 h. After harvesting by centrifugation (6500 g, 15 min,4 °C) and resuspension in 50 mm Hepes, pH 8.0, 300 mmNaCl, purification of the recombinant protein was per-formed as previously described [44]. Fractions containingLipD (11.1 kDa) were identified by 15% SDS–PAGE anal-ysis, pooled, and dialysed against 10 mm Tris ⁄ HCl, pH 8.0,using HiTrap desalting columns (Amersham PharmaciaBiotechnology). Protein concentration was determinedspectrophotometrically using the calculated extinctioncoefficient at 280 nm. The yield of purified protein was2mgÆL)1of culture.In vitro 4¢-phosphopantetheinylation of apo-AcpK andapo-LipD was performed as described above for apo-DptF.In vivo 4¢-phosphopantetheinylation of apo-LipD was car-ried out in BL21(DE3)-pRep4-gsp. The transfer assays toholo-LipD and holo-AcpK and determination of the kineticparameters for the DptE-mediated transfer to holo-LipDwere performed as described above (see Determination ofthe kinetic parameters of DptF lipidation by DptE).AcknowledgementsWe thank Dr Georg Scho¨nafinger, Dr Christoph Mahl-ert and Thomas Knappe (Department of Chemistry ⁄Biochemistry, Philipps-University Marburg, Germany)for helpful discussions and critical comments on themanuscript. Dr Henning D. Mootz provided theHM0079 and HM404 strains. This work was supportedby the Deutsche Forschungsgemeinschaft and the Fondsder Chemischen Industrie.References1 Miao V, Coeffet-Legal MF, Brian P, Brost R, Penn J,Whiting A, Martin S, Ford R, Parr I, Bouchard M et al.(2005) Daptomycin biosynthesis in Streptomyces roseosp-orus: cloning and analysis of the gene cluster and revisionof peptide stereochemistry. Microbiology 151, 1507–1523.2 Hojati Z, Milne C, Harvey B, Gordon L, Borg M, FlettF, Wilkinson B, Sidebottom PJ, Rudd BA, Hayes MAet al. (2002) Structure, biosynthetic origin, and engi-neered biosynthesis of calcium-dependent antibioticsfrom Streptomyces coelicolor. Chem Biol 9 , 1175–1187.3 Fukuda DS, Debono M, Molloy RM & Mynderse JS(1990) A54145, a new lipopeptide antibiotic complex:microbial and chemical modification. J Antibiot 43,601–606.4 Miao V, Brost R, Chapple J, She K, Gal MF &Baltz RH (2006) The lipopeptide antibiotic A54145biosynthetic gene cluster from Streptomyces fradiae.J Ind Microbiol Biotechnol 33, 129–140.5 Heinzelmann E, Berger S, Muller C, Hartner T, PorallaK, Wohlleben W & Schwartz D (2005) An acyl-CoAdehydrogenase is involved in the formation of the Deltacis3 double bond in the acyl residue of the lipopeptideantibiotic friulimicin in Actinoplanes friuliensis. Microbi-ology 151, 1963–1974.6 Vertesy L, Ehlers E, Kogler H, Kurz M, Meiwes J, Sei-bert G, Vogel M & Hammann P (2000) Friulimicins:novel lipopeptide antibiotics with peptidoglycan synthe-sis inhibiting activity from Actinoplanes friuliensis sp.nov. II. Isolation and structural characterization. J Anti-biot 53, 816–827.Lipidation of daptomycin M. Wittmann et al.5352 FEBS Journal 275 (2008) 5343–5354 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... & Shen B (2001) Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide–polyketide antitumor drug bleomycin Chem Biol 8, 725–738 Stein DB, Linne U, Hahn M & Marahiel MA (2006) Impact of epimerization domains on the intermodular transfer of enzyme-bound intermediates in nonribosomal peptide synthesis Chembiochem 7,... Oberthur M & Marahiel MA (2008) Harnessing the chemical activation inherent to carrier protein-bound thioesters for the characterization of lipopeptide fatty acid tailoring enzymes J Am Chem Soc 130, 2656–2666 38 Steller S, Sokoll A, Wilde C, Bernhard F, Franke P & Vater J (2004) Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein Biochemistry 43, 11331–11343 5354 39... Thurston L, Rich P, Miao V & Baltz RH (2006) Complementation of daptomycin dptA and Lipidation of daptomycin 21 22 23 24 25 26 27 28 29 30 31 32 dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics Microbiology 152, 2993–3001 Tang Y, Koppisch AT & Khosla C (2004) The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase... Enzymatic and chemical modifications of lipopeptide antibiotic A21978C: the synthesis and evaluation of daptomycin (LY146032) J Antibiot 41, 1093–1105 11 Schwarzer D, Finking R & Marahiel MA (2003) Nonribosomal peptides: from genes to products Nat Prod Rep 20, 275–287 12 Kopp F & Marahiel MA (2007) Where chemistry meets biology: the chemoenzymatic synthesis of nonribosomal peptides and polyketides Curr Opin... metabolic diversity Curr Opin Struct Biol 17, 736–743 35 Hansen DB, Bumpus SB, Aron ZD, Kelleher NL & Walsh CT (2007) The loading module of mycosubtilin: an adenylation domain with fatty acid selectivity J Am Chem Soc 129, 6366–6367 36 Aron ZD, Fortin PD, Calderone CT & Walsh CT (2007) FenF: servicing the Mycosubtilin synthetase assembly line in trans Chembiochem 8, 613–616 37 Kopp F, Linne U, Oberthur M &... Chain initiation in the leinamycin-producing hybrid nonribosomal peptide ⁄ polyketide synthetase from Streptomyces atroolivaceus S-140 Discrete, monofunctional adenylation enzyme and peptidyl carrier protein that directly load d-alanine J Biol Chem 282, 20273–20282 Hisanaga Y, Ago H, Nakagawa N, Hamada K, Ida K, Yamamoto M, Hori T, Arii Y, Sugahara M, Kuramitsu S et al (2004) Structural basis of the. .. Stubbs MT & Marahiel MA (2007) Aminoacyl-coenzyme A synthesis catalyzed by adenylation domains FEBS Lett 581, 905–910 42 La Clair JJ, Foley TL, Schegg TR, Regan CM & Burkart MD (2004) Manipulation of carrier proteins in antibiotic biosynthesis Chem Biol 11, 195–201 43 Mootz HD, Finking R & Marahiel MA (2001) 4’-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis J Biol Chem... Yabe K (2008) Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis Biosci Biotechnol Biochem 72, 724–734 40 Hitchman TS, Schmidt EW, Trail F, Rarick MD, Linz JE & Townsend CA (2001) Hexanoate synthase, a specialized type I fatty acid synthase in aflatoxin B1 biosynthesis Bioorg Chem 29, 293–307 41 Linne U, Schafer A, Stubbs... (2005) Natural products to drugs: daptomycin and related lipopeptide antibiotics Nat Prod Rep 22, 717–741 8 Kopp F, Grunewald J, Mahlert C & Marahiel MA (2006) Chemoenzymatic design of acidic lipopeptide hybrids: new insights into the structure–activity relationship of daptomycin and A54145 Biochemistry 45, 10474–10481 9 Counter FT, Allen NE, Fukuda DS, Hobbs JN, Ott J, Ensminger PW, Mynderse JS, Preston... the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase Int J Parasitol 37, 307–316 Sauermann R, Rothenburger M, Graninger W & Joukhadar C (2008) Daptomycin: a review 4 years after first approval Pharmacology 81, 79–91 Li H, Melton EM, Quackenbush S, DiRusso CC & Black PN (2007) Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae . Role of DptE and DptF in the lipidation reaction of daptomycin Melanie Wittmann, Uwe Linne, Verena Pohlmann and Mohamed A. MarahielDepartment of Chemistry. dptE and dptF are localized immediatelyupstream of the NRPSs of A21987C. The resultingproteins DptE and DptF were predicted to beinvolved in the lipidation
- Xem thêm -

Xem thêm: Báo cáo khoa học: Role of DptE and DptF in the lipidation reaction of daptomycin ppt, Báo cáo khoa học: Role of DptE and DptF in the lipidation reaction of daptomycin ppt, Báo cáo khoa học: Role of DptE and DptF in the lipidation reaction of daptomycin ppt