TioS T-TE – a prototypical thioesterase responsible for cyclodimerization of the quinoline- and quinoxaline-type class of chromodepsipeptides Lars Robbel, Katharina M. Hoyer and Mohamed A. Marahiel Department of Chemistry, Philipps-University Marburg, Germany Bacteria and fungi of different genera posses a rich arsenal of bioactive compounds to gain evolutionary advantages over competing organisms in their natural habitat. Among such compounds, the class of biologi- cally active peptides represents a rich resource for the discovery of novel pharmaceutical agents. The biosyn- thesis of the oligopeptides can be either carried out via a ribosomal strategy, as in the case of capistruin or patellamide, or via a template-directed manner by multimodular nonribosomal peptide synthetases (NRPSs) [1–3]. Peptides of nonribosomal origin include antitumor compounds (bleomycin), antibiotics (gramicidin S), immunosuppressive agents (cyclospo- rin), biosurfactants (surfactin) and siderophores (bacillibactin) [4–8]. A key structural feature of nonrib- osomally synthesized oligopeptides is their macrocyclic structure, conferring protection against degradation by peptidases and increasing the physico-chemical stability Keywords biocombinatorial synthesis; chromodepsipeptides; iterative cyclodimerization; thiocoraline; thioesterase Correspondence M. A. Marahiel, Department of Chemistry ⁄ Biochemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, D-35043 Marburg, Germany Fax: +49 06421 282 2191 Tel: +49 06421 282 5722 E-mail: marahiel@staff.uni-marburg.de (Received 17 October 2008, revised 11 December 2008, accepted 12 January 2009) doi:10.1111/j.1742-4658.2009.06897.x The family of chromodepsipeptides constitutes a class of structurally related pseudosymmetrical peptidolactones and peptidothiolactones synthe- sized by nonribosomal peptide synthetases. The chromodepsipeptides, which are analogous to the extensively characterized echinomycin, attain their DNA-bisintercalating properties from chromophore moieties attached to the N-termini of the oligopeptide chain. Thiocoraline, a quinoline-substi- tuted DNA-bisintercalator isolated from marine actinomycetes, is a two- fold symmetric octathiodepsipeptide currently undergoing preclinical trials phase II. In the present study, the excised peptide cyclase TioS T-TE (thio- lation-thioesterase bidomain) was employed as a general catalyst for the in vitro generation of thiocoraline analogs. TioS T-TE is capable of catalyz- ing ligation and the subsequent cyclization of tetrapeptidyl-thioester substrates, circumventing the demanding synthesis of octapeptidyl sub- strates. The general importance of several amino acid residues within the tetrapeptide was evaluated and revealed new insights with respect to the iterative mechanism utilized by the thioesterase. Additionally, substrate tolerance towards the cyclizing nucleophile allows the formation of macro- lactones instead of the native macrothiolactones. Several thiocoraline analogs were isolated and investigated for DNA-bisintercalation activity. Relaxed substrate specificity regarding the chromophore moiety enables the chemoenzymatic synthesis of the quinoxaline- and quinoline-type class of chromodepsipeptides. TioS T-TE is the first nonribosomal peptide synthe- tase-derived thioesterase, capable of macrothiolactonization and macrolact- onization, working in an iterative manner. Abbreviations 3HQA, 3-hydroxyquinoline-2-carboxylic acid; IPTG, isopropyl thio-b- D-galactoside; NRPS, nonribosomal peptide synthetase; QA, quinaldic acid; QX, quinoxaline-2-carboxylic acid; SNAC, N-acetylcysteamine; T, thiolation domain; TE, thioesterase domain; t R , retention time. FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 1641 of the cyclic product [9]. Furthermore, the rigidifica- tion of the molecule reduces structural flexibility, leads to the conformation required for interaction with the corresponding target (i.e. receptor proteins or DNA) and ensures biological activity [10]. Macrocyclization is generally mediated by C-termi- nal thioesterase domains (TE, cyclase), located in the termination module of the NRPS assembly line [11]. The resulting structure can be either branched-cyclic, as in the case of the last-line antibiotic daptomycin, or closed, as in the case of the antibiotic tyrocidine A [12,13]. The nature of the intramolecular bond forma- tion catalyzed by the TEs was up to now limited to amide- or ester-linkage giving rise to the corresponding macrolactam or macrolacton. The mechanism of release depends on the type of NRPS, which can be subdivided into three different classes: linear (type A), exemplified by the biosynthesis of tyrocidine A; itera- tive (type B), giving rise to bacillibactin; and nonlinear (type C), as in the case of the iron scavenging sidero- phore coelichelin [8,13–15]. Iterative NRPSs use their modular template more than once to achieve the assembly of the final product from repetitive building blocks [16]. Recently, the iterative thioesterase domain GrsB TE, responsible for the cyclodimerization of pen- tapeptidyl-precursors to form the decapeptide gramici- din S, has been comprehensively analyzed in vitro, providing insights into a unique ligation and ‘head-to- tail’ cyclization mechanism (backward reaction) [17]. Among the iteratively assembled nonribosomal pep- tides, the class of chromodepsipeptides encompasses a broad variety of structurally and functionally diverse compounds (Fig. 1). These peptides are known to bind to duplex DNA through a mechanism known as bisin- tercalation, which is mediated by the twin chromo- phores attached to the macrocyclic molecule [18–20]. Chromodepsipeptides share a common peptidic scaf- fold and a pseudosymmetrical structure as a result of the condensation of two symmetrical halves. Further- more, this class can be subdivided into two main groups (i.e. the quinoxalines and the quinolines), depending on the chromophore moiety bound to the N-termini of each oligopeptide chain. Prominent mem- bers of the quinoxaline-group of chromodepsipeptides N O O N H N O N H O S N N O O N N H O H N O S N O O S S O H H O N O S O N H N O N H S O S N O H N O S O N N H O H N O S N H O O O S N O N H O O O O N O N O O O N O N O H N O N O H N O N O H H O N O O N H N O N H O O N N N O O N N H O H N O O N N O O S M e S N O O N H N O N H O O N N N O O N N H O H N O O N N O O S S N N O N H O O N N O O N O N O O O N O N O H N O N N O H N O N N O H H O R 2 O O R 1 O M e M e O OH HO echinomycin (macrolactone) triostin A (macrolactone) BE-22179 (macrothiolactone) thiocoraline (macrothiolactone) sandramycin (macrolactone)luzopeptine A (macrolactone) disulfide crossbridge thioacetal crossbridge 1 4 2 5 3 6 Q uinoxalines Q uinolines N R 1 = R 2 = COMe N H N H H N O N HO OMe MeO N O N H OH Fig. 1. The class of chromodepsipeptides subdivided into the groups of quinoxalines and quinolines sharing a common peptidic scaffold and a pseudosymmetrical structure. The classification is based on the N-terminally attached chromophore moiety: 1–2, quinoxalines (1, echino- mycin; 2, triostin A); 3-6, quinolines (3, luzopeptine A; 4, thiocoraline; 5, BE-22179; 6, sandramycin). Intramolecular crossbridges are highlighted in grey. TioS T-TE thiocoraline L. Robbel et al. 1642 FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS are echinomycin (antitumor) (1) and triostin A (antitumor) (2), which have been isolated from Strepto- myces echinatus and Streptomyces triostinicus respec- tively [21,22]. These compounds bind specifically to DNA via the insertion of the planar chromophore quinoxaline-2-carboxylic acid (QX), inhibiting tran- scription and replication, which has led to the progres- sion of echinomycin into clinical antitumor trials. The group of quinoline-chromodepsipeptides encompasses the natural products sandramycin (anti-HIV) (6), luzo- peptine A (anti-HIV) (3), BE-22179 (antibiotic) (5) and thiocoraline (antitumor) (4), isolated from Nocardio- ides sp. (ATCC 39419), Actinomadura luzonensis nov. sp., Streptomyces sp. A22179, Micromonospora sp. L13-ACM2-092 and Micromonospora ML1, respec- tively [23–26]. Thiocoraline itself is a two-fold-symmet- ric bicyclic octathiodepsipeptide in which the N-termini of the two oligopeptide chains are capped with the chro- mophore moiety 3-hydroxyquinoline-2-carboxylic acid (3HQA) acting as an intercalating group [26]. The two symmetrical halves consisting of 3HQA-d-Cys1- Gly2-N-methyl-l-Cys3-N,S-dimethyl-l-Cys4 are linked together through two thioester bonds between the N-ter- minal D-Cys1 residue of one half and the N,S-dimethyl- l-Cys4 of the other half. An intramolecular disulfide crossbridge from Cys2 residues leads to a further struc- tural rigidification of this unique macrothiolactone. Thiocoraline shares the d-configured C-terminal amino acid involved in macrocyclization with all known chro- modepsipeptides, whereas the thioester bond is unique to thiocoraline and BE-22179 and represents a novel class of thioesterase-mediated side-chain linkage. Biosynthesis of thiocoraline is carried out by the tetra- modular NRPS assembly line consisting of TioR and TioS, as demonstrated previously [27] (Fig. 2). Due to the fact that the number of amino acids found within the product does not correlate with the total number of adenylation domains, an iterative mechanism of biosynthesis was proposed [27]. Online modifications of the natural amino acids include epimerization of l-Cys1 to d-Cys1 and N-methylation of l-Cys3 ⁄ 4byN-methyltransferase domains integrated into the assembly line. The enzymatic mechanism of S-methylation of l-Cys4 remains to be elucidated. Thiocoraline shows potent anti-bacterial activity against Gram-positive bacteria and a wide spectrum of anti-proliferative activity against various cancer cell lines in vitro that are undergoing preclinical trials phase II [28,29]. Organic synthesis of the aza- and oxa- thiocoraline-class led to compounds with increased physico-chemical stability, potentially increasing the half-life in human plasma from 4 h to clinically appli- cable time spans [30–32]. Recently, a structural basis for the mode of action of thiocoraline has been estab- lished through molecular dynamics simulation of thio- coraline bisintercalating into duplex DNA [33]. Thiocoraline is shown to adopt a U-shaped conforma- tion and to bind to the minor groove of GC-rich sequences, especially those encompassing a central CpG step presumably leading to an inhibition of DNA polymerase a. The planar chromophore moiety 3HQA ensures a tricyclic hydrogen-bonded conformation and facilitates DNA-bisintercalation. The development of in vitro approaches to obtain analogs of thiocoraline TE T C Cys E T C Gly E T C Cys C Cys MM T N HO HN O SH S O N HO HN O SH NH O S O N HO HN O SH NH O N O S O SH AA TE C E T Adenylation domain Condensation domain Epimerisation domain N-Methyltransferase Thioesterase domain Thiolation domain (PCP) N HO HN O SH NH O N O N O SH S HS O 2x TioJ ATP PP I N HO O HO N HO O AMPO TioO N HO O S T Cys E T C Gly E T C Cys C Cys module 1 module 2 module 4module 3 TioR (277.9 kDa) TioS (346.7 kDa) M M T N HO HN O SH S O N HO HN O SH NH O S O N HO HN O SH NH O N O S O SH AA TE C E T N HO HN O SH NH O N O N O SH S HS O 2x TioJ ATP PP I N HO O HO N HO O AMPO TioO N HO O S N HO HN O SH NH O N O N O SH O HS O N O S O N H N O N H S O S N OH N O S O N N H O H N O S N HO O O S thiocoraline TE C M Fig. 2. The tetramodular NRPS assembly line consisting of TioR and TioS. The number of amino acids found in the assembled product does not correlate with the four adenylation domains found in the two peptide synthetases. The iteratively working C-terminal thioesterase medi- ates ligation and subsequent macrothiolactonization of two identical linear chromophore-capped tetrapeptides, as indicated by the blue arrow. L. Robbel et al. TioS T-TE thiocoraline FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 1643 with improved physico-chemical stability and to cir- cumvent low yield organic synthesis is crucial for the generation of potential therapeutic applications based on this class of compounds. In the present study, we report the first in vitro char- acterization of a prototypical thioesterase responsible for the iterative assembly of the quinoline- and quinox- aline-type class of chromodepsipeptides, capable of macrolactonization and macrothiolactonization. The substrate specificity of TioS T-TE was determined and macrocyclization reactions were optimized to obtain maximum yields. Furthermore, the backward mecha- nism proposed for iteratively working thioesterases was confirmed. Chemoenzymatically generated macro- cycles were isolated and investigated for DNA-bisinter- calation activity compared to native thiocoraline. TioS T-TE represents a robust and versatile catalyst for the generation of chromodepsipeptide analogs with a potentially improved spectrum of pharmaceutical properties. Results Expression and isolation of TioS T-TE as active apo-form protein TioS T-TE was heterologously expressed in Escherichia coli M15 ⁄ pREP4 cells at 20 °C and isolated as a C-ter- minally His6-tagged apo-form protein in sufficient yields (8 mgÆL )1 ; see Fig. S1). The inclusion of the adjacent T-domain assured the correct N-terminal fold of the protein. Overall a-helical protein fold, as predicted for a ⁄ b-hydrolases, was confirmed via CD spectropolarimetric analysis (see Fig. S2). Substrate specificity of TioS T-TE To evaluate the biocombinatorial potential and to investigate the combined ligation and macrocyclization mechanism of the excised TE-domain TioS T-TE, a set of tetrapeptidyl-thioesters was synthesized and incubated with the recombinantly generated protein (see Table S1). The sequence of the tetrapeptidyl-sub- strates was initially based on the primary amino acid sequence of the linear thiocoralin tetrapeptidyl-precur- sor. To overcome the lack of synthetically demanding building blocks for solid phase peptide synthesis and to allow the generation of novel thiocoraline analogs, naturally occurring modified amino acids were substi- tuted with commercially available ones. The utilized substrates lacked N-methylation of the C-terminal cystein-residues and the 3-hydroxyfunctionality of the chromophore moiety 3HQA. Stereochemical informa- tion was conserved throughout the oligopeptide chain. For stability reasons, the tetrapeptidyl-substrates were C-terminally activated as N-acetylcysteamines (SNACs) circumventing thiophenol-activation. Fur- thermore, synthetically demanding synthesis of the oc- tapeptidyl-precursors was circumvented by the ligation capability of TioS T-TE, resulting in the utilization of tetrapeptidyl-precursors. All assays were analyzed uti- lizing reversed phase LCMS methods and reported together with the corresponding retention times. Incu- bation of recombinantly produced TioS T-TE with TL1, resembling the most native substrate based on NRPS adenylation-domain specificity prediction, revealed only hydrolytically cleaved linear tetrapeptide. After 1 h, total substrate hydrolysis was detected. This result indicated that the steric demand or the polarity of the C-terminal amino acid is essential for recogni- tion of the substrate and subsequent ligation and macrocyclization. In addition, we speculated that S-methyl-l-Cys4 is incorporated into the oligopeptide chain instead of l-Cys4. This model of biosynthesis would require S-methylation prior to cyclization in vivo. Substitution of l-Cys4 with the sterically more demanding S-methyl-l-Cys4 (TL2) also led to the exclusive formation of hydrolytically cleaved linear tet- rapeptide. Based on these results, l-Cys3 was replaced with l-Ala3 (TL3) to maintain stereochemical infor- mation and to minimize electrostatic repulsion effects between sulfhydrylgroups in close proximity. HPLC- MS analysis of the assay revealed the formation of macrothiolactone Cy3, retention time (t R ) = 27.3 with a hydrolysis (Hy3, t R = 12.1) to cyclization ratio of 12 : 1 and total substrate conversion after 2 h at 25 °C (Fig. 3). Encouraged by the results obtained, and to investigate the mechanism of macrocyclization, the steric demand of the C-terminal amino acid was further increased by the incorporation of l-Met4 (TL4) showing an improved hydrolysis to cyclization ratio of 1 : 2 (Hy4, t R = 14.2; Cy4, t R = 24.7). Addi- tionally, the formation and accumulation of the linear octapeptidyl-SNAC (Lig4, t R = 24.1) was observed representing the main product. In total, the substrate was converted at a ratio of 1 : 4 : 2 (Hy4 ⁄ Lig4 ⁄ Cy4) (Fig. 4). The steric demand of the C-terminal l-Met4 led to the covalent trapping of the ligation product and abol- ished complete macrocyclization. To corroborate the result indicating that l-Cys3 strongly affects macrothi- olactonization, TL5 was synthesized showing a mixed substitution pattern of l-Cys3 and l-Met4. Analogous to the results obtained with TL2, TioS T-TE is not capable of catalyzing ligation or macrothiolactoniza- tion. Total substrate turnover is accomplished after TioS T-TE thiocoraline L. Robbel et al. 1644 FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 2 h, resulting in complete hydrolytic cleavage of the thioester. All chromodepsipeptides share a d-configured amino acid responsible for the nucleophilic attack of the side chain onto the acyl-O-TE oxoester intermediate. To demonstrate the significance of this stereoinformation substrate, TL6 was synthesized harboring l-Cys1 instead of d-Cys1. Using the linear tetrapeptidyl-sub- strate, only hydrolytic cleavage was detected, confirm- ing the necessity of the N-terminal stereogenic center. Biocombinatorial evaluation of TioS T-TE To generate novel chromodepsipeptides with improved physico-chemical stability based on the structure of thiocoraline, an alternative set of subtrates was synthe- sized carrying d-Ser4 as the cyclization-mediating nucleophile instead of d-Cys4. Employment of TL7, the Ser-substituted analog of TL1, showed, in contrast to TL1, macrocylization at a hydrolysis to cyclization ratio of 5 : 1. Products could be assigned, using reversed phase LCMS, to the hydrolysis product (Hy7) macrolactone (Cy7) and a macrolactone with intramo- lecular disulfide connectivity (Cy7SS). Consecutively, TL8 was incubated with TioS T-TE. After 60 min, complete substrate conversion was detected with a hydrolysis (Hy8, t R = 11.2) to cyclization (Cy8, t R = 30.2) ratio of 2 : 1. Additionally, side-product formation could be assigned to a four residue macro- lactone Cy8 ⁄ 4 resulting from an intramolecular attack 20 30 40 50 60 70 80 Retention time ( min ) Absorbance ( 210 nm ) w/o enzyme, 2 h, 37 °C 2 h, 15 °C 2 h, 25 °C 2 h, 37 °C TL3 Cy3 Cy3 Hy3 H y3 H N O S O N H H N O N H O S N N H O S O H N N H O H N O S N O O Cy3 = [M + H + ] = 1007.4 Fig. 3. Cyclization of substrate TL3 medi- ated by TioS T-TE. The HPLC traces corre- spond to the incubation of TL3 (300 l M) with TioS T-TE at specific temperatures for 2 h. The blue HPLC trace corresponds to the control lacking the enzyme at the tem- perature resulting in maximum yields of Cy3. 14 16 18 20 22 24 Absorbance (210 nm) Retention time (min) H N O O N H H N O N H O S N N H O O H N N H O H N O S N O O S S [M + H + ] = 1035.3 Cy4 = TL4 Hy4 Lig4 Cy4 Fig. 4. Cyclization and ligation of substrate TL4 mediated by TioS T-TE. The HPLC traces correspond to the incubation of TL4 (300 l M) with TioS T-TE at 25 °C for 2 h. The blue HPLC trace corresponds to the control lacking the enzyme. L. Robbel et al. TioS T-TE thiocoraline FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 1645 of the side chain nucleophile of d-Ser4 onto the acyl- O-TE oxoester (Cy8 ⁄ 4, t R = 20.5) (see Fig. S3). MS fragmentation studies strongly support the identity of the four residue macrolactone and exclude the forma- tion of an alternative two residue macrothiolactone due to the detection of intense fragments containing dehydro-alanine, which are characteristic for gas-phase fragmentation of lactones (see Doc. S1 and Fig. S4) [34]. Macrocyclization of the linear tetrapeptidyl- SNAC was exclusively limited to substrate TL8 . Substitution of l-Cys3 with l-Ala3 and subsequent incubation of substrate TL9 with TioS T-TE led to a hydrolysis (Hy9, t R = 7.5) to cyclization (Cy9, t R = 27.5) ratio of 8 : 1 at 25 °C (Fig. 5). Based on the results obtained with TL4, the analogous substrate TL10 was synthesized. HPLC-MS analysis revealed the formation of the macrocycle Cy10 at a hydrolysis to cyclization ratio of 8 : 1. Additionally, the formation of the linear octapeptidyl-SNAC Lig10 was detected, reflecting the steric demand of l-Met4. To investigate the influence of the chromophore moiety on cycliza- tion-efficiency, and to establish TioS T-TE as a general catalyst for the ligation and cyclization of the quino- line- and quinoxaline-type class of chromodepsipep- tides, TL11 was synthesized. The primary sequence was based on TL3 with the exception of the chromo- phore moiety quinaldic acid (QA), which was sub- stituted with QX, the chromophore found in echinomycin and triostin A. The cyclization reaction profile revealed a hydrolysis to cyclization ratio of 8 : 1 in analogy to substrate conversion of TL3. Sub- strate conversion was completed after 1 h of incuba- tion at 25 °C. This result indicates the general relaxed substrate specificity of the cyclase towards the N-termi- nal chromophore and implies that TioS T-TE can serve as a prototypical TE for the assembly of quino- line or quinoxaline carrying compounds. Temperature dependence of macrocyclization To improve the cyclization yields and to decrease the hydrolytic release of the linear peptidyl-precursor, the substrates TL3 and TL9, both differing only in the nature of the cyclization-mediating nucleophile (d-Cys4 or d-Ser4) were employed in assays at varying temperatures. The temperatures chosen were 15, 25 and 37 °C respectively. TL3 showed an improved hydrolysis to cyclization ratio of 3 : 1 at 15 °C com- pared to a ratio of 12 : 1 (Hy3 ⁄ Cy3)at25°C. The best macrothiolactone (Cy3, t R = 27.3) yields were obtained at 37 °C with an altered reaction profile revealing a low flux towards hydrolysis (Hy3, t R = 12.1) and a shifted hydrolysis to cyclization ratio of 1 : 7 (Fig. 3). Kinetic parameters were determined for total substrate conversion at 37 °C revealing a k cat of 5.26 ± 0.64 min )1 . By contrast, TL9 was cyclized more efficiently at low temperatures. An improved hydrolysis to cyclization ratio of 4 : 1 was observed at 15 °C compared to a ratio of 8 : 1 at 25 °C(Hy9, t R = 7.5; Cy9, t R = 27.3) (Fig. 5). Interestingly cycli- zation was completely abolished at 37 °C. Only the formation of the linear tetrapeptide (Hy9) was detected (Fig. 5). Additionally, substrate TL8 was examined towards temperature dependence of macrolactoniza- tion. Best macrocyclization yields were obtained at 15 °C with a hydrolysis to cyclization ratio of 1 : 4 compared to a ratio of 2 : 1 at 25 °C(Hy8, t R = 11.2; Cy8, t R = 30.2). At 37 °C, cyclization yields were reduced, consistent with the results obtained with TL9, to a ratio of 9 : 1 towards hydrolysis (see Fig. S3). 10 20 30 40 50 60 70 w/o enzyme, 2 h, 37 °C 2 h, 15 °C 2 h, 25 °C 2 h, 37 °C Retention time ( min ) Absorbance ( 210 nm ) Cy9 Cy9 Hy9 Hy9 TL9 TL9 Hy9 TL9 H N O S O N H H N O N H O O N N H O S O H N N H O H N O O N O O Cy9 = [M + H + ] = 975.4 Fig. 5. Cyclization of substrate TL9 medi- ated by TioS T-TE. The HPLC traces corre- spond to the incubation of TL9 (300 l M) with TioS T-TE at specific temperatures for 2 h. The blue HPLC trace corresponds to the control lacking the enzyme at the tem- perature resulting in maximum yields of Cy9. TioS T-TE thiocoraline L. Robbel et al. 1646 FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS Kinetic parameters were determined for TL8 at 15 °C resulting in a k cat of 8.92 ± 1.2 min )1 . An overview of hydrolysis to cyclization ratios is given for the investi- gated substrates in graphical form in Fig. S5A–C. DNA-bisintercalation activity assay To evaluate the DNA-bisintercalative properties of chemoenzymatically generated thiocoraline analogs and to elucidate structural features contributing to DNA-insertion, four tetrapeptidyl thioesters were syn- thesized. In accordance with the previously discussed results, the macrocyclization assays were carried out under optimal conditions. Isolation of the correspond- ing macrocyles (Cy3 ⁄ Cy8 ⁄ Cy8SS ⁄ Cy9 ⁄ Cy11) was achieved by HPLC separation. (Fig. 6). To compare the bisintercalation capability of the novel analogs, native thiocoraline was also isolated and subjected to the DNA-melting assay. The sequence of the utilized oligonucleotide was based on results obtained previ- ously [33]. Incubation of the oligonucleotide AT with thiocoraline and a subsequent DNA-melting experi- ment resulted in a melting curve demonstrating a hys- teresis shape characteristic of DNA-bisintercalators. The duplex DNA was stabilized by 15.9 °C [33]. Incu- bation of the same oligonucleotide with the isolated macrolactones and macrothiolactones led to a marginal stabilization of 0.1–0.2 °C (data not shown). Discussion The exploitation of the macrocyclization potential inherent in TEs dissected from their corresponding nonribosomal peptide synthetases has enabled the gen- eration of novel macrocyclic bioactive compounds, based on the primary sequence of the native substrate, under stringent stereo- and regioselective control. Among the class of nonribosomally synthesized pep- tides, the chromodepsipeptides represent a multitude of structurally and functionally diverse compounds. With the comprehensive biochemical characterization of TioS T-TE, we have established a model system for the biocombinatorial synthesis of the quinoline- and quinoxaline-type class of chromodepsipeptides. By con- trast to linearly operating TEs, TioS T-TE acts as an iterative ligation and macrocyclization platform that is capable of catalyzing macrolactonization and a so far unreported macrothiolactonization. Initially, TioS T-TE was tested using a linear tetra- peptide based on the amino acid sequence derived from the specificity prediction of the corresponding adenylation domains. Incubation of TL1 with the thioesterase resulted solely in hydrolytic cleavage of the C-terminally SNAC activated thioester. This result led to the conclusion that the steric demand of the C-terminal amino acid is crucial for suppression of hydrolysis by shielding the acyl-O-TE oxoester inter- mediate from the nucleophilic attack of water. Pre- suming that S-methylation of the naturally occurring S-methyl-l-Cys4 is carried out prior to recognition, activation and incorporation of the building block into the oligopeptide chain, TL2 was synthesized and employed in the macrocyclization assay. Under these conditions, hydrolysis was reduced, with little sub- strate remaining after 2 h of incubation, in contrast to total substrate conversion in the case of TL1, confirming the assumption made concerning hydroly- sis suppression by steric demand. To evaluate the H N O S O N H H N O N H O O N N H O S O H N N H O H N O O N O O S H H S H N O S O N H H N O N H O O N N H O S O H N N H O H N O O N O O S S H N O S O N H H N O N H O S N N H O S O H N N H O H N O S N O O H N O S O N H H N O N H O O N N H O S O H N N H O H N O O N O O H N O S O N H H N O N H O S N N N H O S O H N N H O H N O S N N O O Cy8 Cy8SS C y 11 Cy3 Cy9 Fig. 6. Isolated macrocycles for the analysis of DNA-bisintercalation properties. Product identities were confirmed by ESI-MS and stabilization of duplex DNA was compared with native thiocoraline 4. L. Robbel et al. TioS T-TE thiocoraline FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 1647 influence of the cysteine residue, forming the disulfide crossbridge, on macrothiolactonization, TL3 was employed harboring a l-Ala3 residue to maintain ste- reochemical information and, concurrently, to reduce the electrostatic repulsion effects of two neighboring sulfhydryl groups. Detection of the macrocyclic prod- uct indicated a strong influence of this position on the ligation and cyclization reaction. In the assembled native thiocoraline, the sulfhydryl groups of l-Cys3 form a disulfide crossbridge minimizing conforma- tional freedom to a great extent. It can be assumed that the oxidative formation of the crossbridge is car- ried out on the T-bound linear octapeptidyl-thioester resulting in a prefold facilitating subsequent macro- cyclization. This assumption is in compliance with the backward mechanism proposed for iteratively working thioesterases, where the T-domain serves as a holding bay for the dimerized product. In the case of echino- mycin biosynthesis, an oxidoreductase (Ecm17) is found within the biosynthetic operon proposed to be responsible for disulfide formation [35]. This oxidore- ductase, although lacking in the gene cluster enabling thiocoraline biosynthesis, could carry out the online modification of the linear T-bound octapeptide in trans [27]. To further demonstrate that the steric demand of the C-terminus is a key position in thio- coraline macrothiolactonization, S-methyl-l-Cys4 was substituted with l-Met4, resulting in an improved hydrolysis to cyclization ratio of 1 : 2 (TL3,12:1) reinforcing former presumptions. Intriguingly, the substitution also led to the buildup of a linear liga- tion product that can be directly assigned to the backward mechanism. The TE-bound tetrapeptide underwent a nucleophilic attack by the external tetrapeptidyl-SNAC mimicking the T-bound tetra- peptide. The C-terminal steric demand inhibited sub- sequent macrocylization and led to the accumulation of the octapeptidyl-SNAC. These observations directly correlate with the results obtained with GrsB T-TE [17]. All known chromodepsipeptides share a d-con- figured N-terminal amino acid that harbors the nucleophilic side-chain mediating cyclization [18]. Substitution of this position with l-Cys1 (TL6) abol- ished ligation and subsequent cyclization resulting in complete hydrolysis. This observation indicates that only d-configured amino acids enable the specific angle, following the Bu ¨ rgi–Dunitz trajectory, required for the nucleophilic attack onto the acyl-O-TE oxo- ester intermediate [36]. Furthermore, the correct posi- tioning of the substrate within the catalytic pocket of the thioesterase might be influenced. To further investigate the biocombinatorial potential of TioS T-TE, a set of d-Ser1 substituted tetrapept- idyl-SNACs (TL7-TL10) was tested. By contrast to TL1, the Ser-substituted TL7 was cyclized leading to the conclusion that only l-Cys3 influences macrothiol- actonization. In this case, the electrostatic repulsion effects only occur when l-Cys3 of one half and d-Cys4 of the other peptide chain are in close proximity. With the sterically less demanding d-Ser4 macrolactoniza- tion is feasible even in presence of l-Cys2. When incu- bating TioS T-TE with TL8, the formation of a four residue macrolactone (Cy8 ⁄ 4) was detected. This mac- rolactonization of a single tetrapeptidyl-SNAC was only observed with TL8. By contrast to TL7, the C-terminal steric demand leads to a more stable TE- bound intermediate, allowing an intramolecular attack of d-Ser1 onto the acyl-O-TE oxoester intermediate prior to hydrolytic cleavage. Alteration of the C-termi- nal chromophore from quinoline to quinoxaline did not influence the cyclization yields to a great extent and allows the generation of quinoxaline-type chro- modepsipeptides. Furthermore, TioS T-TE is the first dissected cyclase catalyzing both macrothiolactoniza- tion and macrolactonization. Enzymatic peptide cyclization often displays low effi- ciency due to the occurrence of hydrolysis of the acyl- O-TE oxoester intermediate. Previous studies on the excised TE-domains from tyrocidine and pristinamycin synthetases revealed hydrolysis to cyclization ratios of 1 : 1 and 1 : 3 for natural substrate analogs [37,38]. The macrocyclization assays described in the present study also revealed a high degree of hydrolysis typical for some isolated TE-domains. To improve cyclization yields, the temperature dependence of either macrothi- olactonization or macrolactonization was evaluated. TE-mediated macrothiolactonization represents an energetically less favored reaction due to the fact that a thermodynamically stable oxoester is converted to a high energy thioester. Increasing the temperature also increased the forma- tion of the endergonically generated macrothiolactone in the case of TL3 and TL11. A reduction of the tem- perature also resulted in the increase of cyclization yields. We speculate that low temperatures lead to a more compact conformation of the enzyme. Under these conditions, premature hydrolysis is reduced, increasing the stability of the acyl-O-TE oxoester inter- mediate that is capable of reacting with further mole- cules to give rise to the macrocyle. By contrast, the thermodynamically indifferent macrolactonization is favored at low temperatures utilizing TL9. Analogous results were obtained with substrate TL8. In all exam- ined cases, total substrate conversion is decelerated at lower temperatures, reflecting the minimized reaction velocities. Kinetic investigation of TL3 turnover TioS T-TE thiocoraline L. Robbel et al. 1648 FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS resulted in a k cat of 5.26 ± 0.64 min )1 , which is in the range of the corresponding substrate turnover of the linear pentapeptidyl-thiophenol of GrsB T-TE (k cat = 2.4 min )1 ). The substrate turnover of TL8 is given by a k cat of 8.92 ± 1.2 min )1 . The higher k cat - value for TL8 is result of an increased flux towards hydrolysis compared to TL3 and does not mean an improved cyclization efficiency. Higher catalytic effi- ciencies can be expected when the linear octapeptide is used due to the ligation reaction comprising the rate- determining step, as described for GrsB T-TE [17]. Recently, Oikawa et al. have shown an alternative improvement method for the cyclodimerization reac- tion of triostin A analogs [38a]. Coincubation with DNA led to the suppression of product inhibition and hydrolysis by exploiting the DNA-bisintercalative properties of the compounds. The mechanisms of how iteratively operating thioes- terases can control the number of repetitive ligation steps remain unknown. Throughout all cyclization reactions, the ring size of the resulting macrocycles was limited to a four residue ring (Cy8 ⁄ 4) or to eight residue rings. By contrast, GrsB T-TE is capable of trimerizing pentapeptidyl-SNAC substrates to form 15-residue rings. It was suggested that the size of the resulting ring and the preorganization of the substrate determine whether a ligation or a cyclization step is carried out [39]. Unfortunately, the prefold of the lin- ear thiocoraline octapeptide has not been investigated. In addition, 12-residue rings could exceed the maxi- mum capacity of the catalytic pocket. To investigate the potential bioactivity of the generated macrocycles, several thiocoraline analogs were isolated and employed in a DNA-bisintercalation activity assay. Authentic thiocoraline stabilized duplex DNA in a range similar to the results described previously, whereas bisintercalation of the analogs could not be detected [33]. The generated thiocoraline analogs dis- played a variety of modifications of the peptidic back- bone compared to the native bisintercalator. Substitution of the naturally occurring chromophore moiety 3HQA with QA or QX is unlikely to affect bisintercalation properties. QX is found in the well characterized DNA-bisintercalators echinomycin and triostin A; nervertheless, Cy11 did not show any activ- ity. Furthermore, QA-substituted chromodepsipeptides, belonging to the recently synthesized FAJANU peptide family, also showed bioactivity against several tumor cell lines [40]. FAJANU 7, a QA-capped eight residue macrolactam, displayed the highest bioactivity, exceed- ing 3HQA or QX harboring compounds. The lack of N-methylation of l-Cys3 ⁄ 4 is presumably responsible for the absence of DNA-bisintercalation activity. N-methylation induces conformational changes and elevates rotational barriers [41]. This rigidification of molecular dynamics gives rise to a preferential prefold of the oligopeptide. The substitution of N-methyl-Gly residues with Gly in the case of FAJANU chromodep- sipeptides led to a decrease of bioactivity by one order. Obviously, additional extensive studies will be neces- sary to gain further insights into the molecular mecha- nism of thiocoraline bioactivity. In conclusion, the excised thioesterase of thiocora- line is a versatile catalyst for the in vitro generation of chromodepsipeptide analogs. TioS T-TE is the first cyclase to be characterized that is capable of catalyzing macrothiolactonization. Additionally, macrolactoniza- tion is feasible due to relaxed substrate specificity towards the cyclizing nucleophile. By utilizing opti- mized assay conditions, cyclization yields can be improved by temperature shifts. Substrate tolerance towards the chromophore moiety also allows the chemoenzymatic synthesis of quinoxaline substituted analogs mimicking the class of triostins and echino- mycins. The approach described in the present study provides new opportunities for developing novel com- pounds related to thiocoraline and similar oligopep- tides with a potentially improved spectrum of pharmaceutical properties and higher in vivo stability. Experimental procedures Bacterial strains, plasmids, biochemicals, chemicals and general methods E. coli Top10 was used as general host for subcloning and E. coli M15 ⁄ pREP4 (Qiagen, Hilden, Germany) was used as host for heterologous expression of TioS T-TE. The thiocoraline producing strain Micromonospora sp. L13- ACM2-092 (CECT-3326) was purchased from the Spanish Type Culture Collection (CECT, University of Valencia, Valencia, Spain). The expression vector pQE60 (Qiagen) was originally from commercial sources. Oligonucleotides were purchased (Operon, Cologne, Germany). Orthogonally protected amino acids were purchased from Novabiochem (Bad Soden, Germany), Bachem Biosciences (Weil am Rhein, Germany) and Anaspec (San Jose, CA, USA). All other compounds except HBTU and HOBt (IRIS Biotech, Marktredwitz, Germany) were purchased from Sigma- Aldrich (Munich, Germany). Standard protocols were applied for all DNA manipulations [42]. Cloning and expression of TioS T-TE The tioS T-TE fragment was synthesized by EZBiolabs (Westfield, IN, USA) including an optimization of codon L. Robbel et al. TioS T-TE thiocoraline FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS 1649 bias for heterologous expression in E. coli. The plasmid pBluescriptIISK(+) carrying the target gene was digested with BamHI and NcoI, and the resulting gene fragment subsequently ligated into a BamHI and NcoI-digested pQE60 vector (Qiagen), appending an C-terminal hexahisti- dine tag to the expressed protein. DNA sequencing of the derived plasmid was performed by GATC Biotech (Kon- stanz, Germany) on an ABIprism 310 genetic analyzer (Applied Biosystems, Carlsbad, CA, USA). For heterolo- gous expression, the plasmid was transformed into E. coli M15 ⁄ pREP4 (Qiagen) cells via electroporation. The trans- formed cells were grown until D 600 of 0.6 was reached at 37 °C, induced with 0.1 mm isopropyl thio-b-d-galactoside (IPTG) and cultivated for an additional 5 h at 20 °C. The heterologously produced protein was purified by Ni-NTA affinity chromatography (Amersham Pharmacia Biotech, Munich, Germany). Fractions containing the protein were identified via SDS-PAGE. Dialysis into 25 m m Hepes and 50 mm NaCl (pH = 6.0) was carried out using HiTrap des- alting columns (GEHealthcare, Munich, Germany). The concentration of the purified protein was determined spec- trophotometrically using the estimated extinction coefficient at 280 nm. After being flash-frozen in liquid nitrogen, the protein was stored at –80 °C. CD spectropolarimetry CD spectra were carried out on a J-810 spectropolarimeter (Jasco, Groß-Umstadt, Germany) at a final concentration of 5 lm TioS T-TE at 20 °C with 20 nmÆmin )1 and a response of 2 s in 10 mm Na 2 HPO 4 buffer (pH = 7.00). Synthesis of the linear tetrapeptidyl-thioester substrates All linear tetrapeptides were produced by solid phase pep- tide synthesis on an APEX 396 synthesizer (Advanced ChemTech, Gießen, Germany) (0.1 mmol scale) with 2-chlorotrityl resin as solid support (IRIS Biotech). The preparation of the C-terminally SNAC-activated peptides was carried out under the utilization of established proto- cols [43]. The identities of the peptidyl-SNAC substrates were determined by reversed phase LCMS (Agilent 1100 MSD) (Agilent, Waldbronn, Germany) (see Table S1). Pep- tides were solubilized in dimethylsulfoxide to a final con- centration of 20 mm and stored at )20 °C. Enzymatic assays Enzymatic assays were carried out in a total volume of 50 lL in assay buffer (25 mm Hepes, 50 mm NaCl, pH = 6.0) at 25 °C. For temperature dependence evalua- tion of macrocycle formation, the temperature was altered to 15 or 37 °C respectively. The final concentration of sub- strate was 300 lm and the total concentration of dimethyl- sulfoxide was 8% (v ⁄ v). The assay was initiated by the addition of 10 lm TioS T-TE and quenched after 2 h by the addition of 10 lL4%(v⁄ v) trifluoroacetic acid. Reduction of oxidatively formed disulfide-bonds was accomplished by the addition of 300 lm Tris-(carboxyeth- yl)-phosphine in dimethylsulfoxide. Assays were analyzed by reversed phase LCMS (Agilent 1100 MSD) on a Nucleo- dur 125 ⁄ 2C 8 (ec) column (pore diameter 100 A ˚ ; particle size 3 lm; Macherey and Nagel, Du ¨ ren, Germany) utilizing the following solvent gradient: 0–50 min, 20–80% MeCN ⁄ 0.1% trifluoroacetic acid into H 2 O ⁄ 0.1% trifluoroacetic acid, 50–55 min, 80–95% MeCN ⁄ 0.1% trifluoroacetic acid into H 2 O ⁄ 0.1% trifluoroacetic acid, 0.2 mLÆmin )1 ,45°C. Identi- ties of the products were confirmed by ESI-MS (Table 1). Kinetics of total substrate turnover were performed by determining the initial conversion rates of nine substrate concentrations, using three time points at each concentra- tion within the linear region of the reaction. The concentra- tion of linear tetrapeptidyl-thioester substrates was Table 1. ESI-MS characterization of linear and cyclic products. Hydrolysis to cyclization ratios are given for the optimal cyclization conditions. Compound Sequence Species (m ⁄ z) Lig observed mass (calculated mass) Cy observed mass (calculated mass) Hydrolysis : cyclization ratio TL1 QA- D-Cys1-Gly2-L-Cys3-L-Cys4-SNAC [M+H] + – (1162.2) – (1043.2) ⁄ TL2 QA- D-Cys1-Gly2-L-Cys3-S-methyl-L-Cys4-SNAC [M+H] + – (1190.2) – (1071.2) ⁄ TL3 QA- D-Cys1-Gly2-L-Ala3-L-S-methyl-L-Cys4-SNAC [M+H] + – (1126.3) 1007.4 (1007.3) 1 : 7 TL4 QA- D-Cys1-Gly2-L-Ala3-L-Met4-SNAC [M+H] + 1154.4 (1154.3) 1035.3 (1035.3) 1 : 2 TL5 QA- D-Cys1-Gly2-L-Cys3-L-Met4-SNAC [M+H] + – (1218.3) – (1099.2) ⁄ TL6 QA- L-Cys1-Gly2-L-Ala3-L-Met4-SNAC [M+H] + – (1154.3) – (1035.3) ⁄ TL7 QA- D-Ser1-Gly2-L-Cys3-L-Cys4-SNAC [M+H] + – (1130.3) 1011.3 (1011.2) 5 : 1 TL8 QA- D-Ser1-Gly2-L-Cys3-L-S-methyl-L-Cys4-SNAC [M+H] + – (1158.3) 1039.1 (1039.3) 1 : 4 TL9 QA- D-Ser1-Gly2-L-Ala3-S-methyl-L-Cys4-SNAC [M+H] + – (1094.4) 975.4 (975.3) 4 : 1 TL10 QA- D-Ser1-Gly2-L-Ala3-L-Met4-SNAC [M+H] + 1121.4 (1121.4) 1002.3 (1002.3) 8 : 1 TL11 QX- D-Cys1-Gly2-L-Ala3-S-methyl-L-Cys4-SNAC [M+H] + – (1128.3) 1009.3 (1009.3) 8 : 1 TioS T-TE thiocoraline L. Robbel et al. 1650 FEBS Journal 276 (2009) 1641–1653 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug Invest New Drugs 22, 24 1–2 51 ´ 33 Negri A, Marco E, Garcı´ a- Hernandez V, Domingo A, ´ Llamas-Saiz AL, Porto-Sanda S, Riguera R, Laine W, David-Cordonnier MH, Bailly C et al (2007) Antitumor activity, X-ray crystal structure, and DNA binding properties of thiocoraline A, a natural bisintercalating thiodepsipeptide... rate of 1 °CÆmin)1 to a final temperature of 95 °C Consecutively, a reverse experiment was conducted by decreasing the temperature form 95 to 25 °C utilizing the same temperature gradient Throughout the process, absorption at 260 nm was measured The midpoint of the transition (Tm) was calculated and compared with a control lacking the bisintercalator On the basis of these results, the stabilization of. .. (Agilent 1100 FLD; excitation 365 nm, emission 540 nm) according to established protocols The retention time of thiocoraline was 42.3 min Total yield of thiocoraline was 1.6 mgÆL)1 culture TioS T-TE thiocoraline DNA-bisintercalation activity assay Oligonucleotides AS1 5¢-AATATACGTTCGATTAA-3¢ and AS2 3¢-TTATATGCAAGCTAATT-5¢ were synthesized by Operon on a 50 nm scale Annealing of each 5¢-oligonucleotide... M, Miyaki T, Koshiyama H & Kawaguchi H (1980) BBM-928, a new antitumor antibiotic complex I Production, isolation, characterization and antitumor activity J Antibiot 33, 108 7–1 097 Okada H, Suzuki H, Yoshinari T, Arakawa H, Okura A, Suda H, Yamada A & Uemura D (1994) A new topoisomerase II inhibitor, BE-22179, produced by a streptomycete I Producing strain, fermentation, isolation and biological activity... structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264 J Am Chem Soc 130, 1144 6–1 1454 2 Schmidt EW, Nelson JT, Rasko DA, Sudek S, Eisen JA, Haygood MG & Ravel J (2005) Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella Proc Natl Acad Sci USA 102, 731 5–7 320... complementary 3¢-oligonucleotide at a final duplex concentration of 2 lm in 10 lL phosphate buffer (10 mm sodiumphosphate, 100 mm NaCl, pH = 7.0) was accomplished in a standard thermocycler (Thermocycler personal; Eppendorf) by heating the solution to 95 °C for 5 min and gradually cooling to 20 °C at a rate of 1 °CÆmin)1 Samples were incubated with authentic thiocoraline or the isolated macrocycles at a final... Albericio F (2006) Total solid-phase synthesis of the azathiocoraline class of symmetric bicyclic peptides Chemistry 12, 900 1–9 009 ´ 31 Tulla-Puche J, Bayo-Puxan N, Moreno JA, Francesch AM, Cuevas C, Alvarez M & Albericio F (2007) Solidphase synthesis of oxathiocoraline by a key intermolecular disulfide dimer J Am Chem Soc 129, 532 2–5 323 32 Brandon EF, Sparidans RW, Meijerman I, Manzanares I, Beijnen JH... functional interactions between nonribosomal peptide synthetases and a polyketide synthase Chem Biol 7, 62 3–6 42 5 Kratzschmar J, Krause M & Marahiel MA (1989) Gramicidin S biosynthesis operon containing the structural genes GrsA and GrsB has an open reading frame encoding a protein homologous to fatty acid thioesterases J Bacteriol 171, 542 2–5 429 6 Zocher R, Nihira T, Paul E, Madry N, Peeters H, Kleinkauf... activity J Antibiot 47, 12 9– 135 Perez BJ, Canedo LM & Fernandez-Puentes JL (1997) Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine Micromonospora II Physicochemical properties and structure determination J Antibiot 50, 73 8–7 41 Lombo F, Velasco A, Castro A, de la Calle F, Brana AF, Sanchez-Puelles JM, Mendez C & Salas JA (2006) Deciphering the biosynthesis pathway of the antitumor... anti-tumor activity Br J Cancer 80, 97 1–9 80 29 Romero F, Espliego F, Baz JP, de Quesada TG, Gravalos D, de laCalle F & Fernandez-Puentes JL (1997) Thiocoraline, a new depsipeptide with antitumor activity produced by a marine Micromonospora I Taxonomy, fermentation, isolation, and biological activities J Antibiot 50, 73 4–7 37 ´ ´ 30 Bayo-Puxan N, Fernandez A, Tulla-Puche J, Riego E, Cuevas C, Alvarez M & Albericio . ligation and macrocyclization platform that is capable of catalyzing macrolactonization and a so far unreported macrothiolactonization. Initially, TioS T-TE. activity assay Oligonucleotides AS1 5¢-AATATACGTTCGATTAA-3¢ and AS2 3¢-TTATATGCAAGCTAATT-5¢ were synthe- sized by Operon on a 50 nm scale. Annealing of each