Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase

Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.     

Stereospecificity of ketoreductase domains 1 and 2 of the tylactone modular polyketide synthase

Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.     

An Iterative Module in the Azalomycin F Polyketide Synthase Contains a Switchable Enoylreductase Domain

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension‐module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be “toggled” off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly‐line catalysis and may explain examples of apparent non‐colinearity in other modular PKS systems.     

An Iterative Module in the Azalomycin F Polyketide Synthase Contains a Switchable Enoylreductase Domain

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be “toggled” off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems.     

Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ～3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.     

Total syntheses of epothilones B and d

A convergent, total synthesis of epothilones B (2) and D (4) is described. The key steps are Normant coupling to establish the desired (Z)-stereochemistry at C12-C13, Wadsworth-Emmons olefination of methyl ketone 28 with the phosphonate ester 8, diastereoselective aldol condensation of aldehyde 5 with the enolate of keto acid derivatives to form the C6-C7 bond, selective deprotection of acid 52, and macrolactonization.     

Mechanisms of molecular recognition in the pikromycin polyketide synthase

BACKGROUND: Modular polyketide synthases (PKSs) produce a wide range of medically significant compounds. In the case of the pikromycin PKS of Streptomyces venezuelae, four separate polypeptides (PikAI-PikAIV), comprising a total of one loading domain and six extension modules, generate the 14-membered ring macrolactone narbonolide. The polypeptide PikAIV contains a thioesterase (TE) domain and is responsible for catalyzing both the last elongation step with methylmalonyl CoA, and subsequent release of the final polyketide chain elongation intermediate from the PKS. Under certain growth conditions this polypeptide is synthesized from an alternative translational start site, giving rise to an N-terminal truncated form of PikAIV, containing only half of the ketosynthase (KS(6)) domain. The truncated form of PikAIV is unable to catalyze the final elongation step, but is able to cleave a polyketide chain from the preceding module on PikAIII (ACP(5)), giving rise to the 12-membered ring product 10-deoxymethynolide. RESULTS: S. venezuelae mutants expressing hybrid PikAIV polypeptides containing acyl carrier protein (ACP) and malonyl CoA specific acyltransferase (AT) domains from the rapamycin PKS were unable to catalyze production of 12- or 14-membered ring macrolactone products. Plasmid-based expression of a hybrid PikAIV containing the native KS(6) and TE domains, however, restored production of both narbonolide and 10-deoxymethynolide in the S. venezuelae AX912 mutant that generates a TE-deleted form of PikAIV. Use of alternative KS domains or deletion of the KS(6) domain within the hybrid PikAIV resulted in loss of both products. Plasmid-based expression of a TE domain of PikAIV as a separate polypeptide in the AX912 mutant resulted in greater than 50% restoration of 10-deoxymethynolide, but not in mutants expressing a hybrid PikAIV bearing an unnatural AT domain. Mutants expressing hybrid PikAIV polypeptides containing the natural AT(6) domains and different ACP domains efficiently produced polyketide products, but with a significantly higher 10-deoxymethynolide/narbonolide ratio than observed with native PikAIV. CONCLUSIONS: Dimerization of KS(6) modules allows in vivo formation of a PKS heterodimer using PikAIV polypeptides containing different AT and ACP domains. In such heterodimers, the TE domain and the AT(6) domain responsible for formation of the narbonolide product are located on different polypeptide chains. The AT(6) domain of PikAIV plays an important role in facilitating TE-catalyzed chain termination (10-deoxymethynolide formation) at the proceeding module in PikAIII. The pikromycin PKS can tolerate the presence of multiple forms (active and inactive) of PikAIV, and decreased efficiency of elongation by PikAIV can result in increased levels of 10-deoxymethynolide. These results provide new insight into functional molecular interactions and interdomain recognition in modular PKSs.     

Synthesis, structure proof, and biological activity of epothilone cyclopropanes

[structure--see text] A semisynthetic route to epothilone cyclopropanes from epothilones A and B is described. Of significance, the deoxygenation of the 12, 13-epoxide to give the corresponding olefin was achieved with high efficiency. The title compounds (8, 9) were active in both tubulin polymerization and cytotoxicity assays, which is in direct contrast to a previously published report. These results provide further evidence that the role of the 12,13-epoxide of epothilones is largely conformational and argue against some of the current pharmacophore models.     

Complex target-oriented total synthesis in the drug discovery process: the discovery of a highly promising family of second generation epothilones

The total synthesis of a family of (E)-9,10-dehydro derivatives of epothilone D (i.e., 12,13-desoxyepothilone B) is described. The route is particularly concise and amenable to production of new congeners. Furthermore, the chemistry described herein constitutes a major simplification in the total synthesis of EpoD, which is in human clinical trials. This new family of epothilones shows major advantages in terms of their potency and pharmacostability relative to the wild-type saturated analogues in the D series. From the perspective of compound availability through synthesis, potency, and pharmacokinetic properties, these compounds could well warrant advancement to clinical evaluation in humans.     

cis-Delta(2,3)-double bond of phoslactomycins is generated by a post-PKS tailoring enzyme

The antifungal phoslactomycins (PLM A-F), produced by Streptomyces sp. HK803, are structurally unusual in that three of their four double bonds are in the cis form (Delta12,13, Delta14,15, Delta2,3). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1, 2, and 5 required for establishing two of these cis double bonds (Delta12,13, Delta14,15), as well as the only trans Delta6,7 double bond. By contrast, the formation of the cis Delta2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Delta2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained are made in plmT2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Delta2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT2 deletion mutant can be accomplished by plasmid based expression of plmT2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydratase-independent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated.     

A Long‐Range Acting Dehydratase Domain as the Missing Link for C17‐Dehydration in Iso‐Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso‐migrastatin (iso‐MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module‐6, ‐9, ‐10 of MgsF (i.e., DH6, DH9, DH10) and module‐11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso‐MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β‐hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long‐range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

Synthesis,Profiling, and Bioactive Conformation of trans‐Cyclopropyl Epothilones

A series of new 3‐deoxy‐C(12),C(13)‐trans‐cyclopropyl‐epothilones have been prepared, bearing benzothiazole, quinoline, thiazol‐5‐ylvinyl, or isoxazol‐3‐ylvinyl side chains. For analogs with fused aromatic side chains, macrocyclic ring‐closure was based on ring‐closing olefin metathesis (RCM) of a precursor incorporating the fully elaborated heavy atom framework of the target structure (including the side chain moiety), while side chain attachment for the thiazole and isoxazole‐containing 16‐desmethyl analogs was performed only after establishment of the macrolactone core. Two approaches were elaborated for a macrocyclic aldehyde as the common precursor for the latter analogs that involved ring‐closure either by RCM or by macrolactonization. Benzothiazole‐ and quinoline‐based analogs were found to be highly potent antiproliferative agents; the two analogs with a thiazol‐5‐ylvinyl or an isoxazol‐3‐ylvinyl side chain likewise showed good antiproliferative activity but were significantly less potent than the parent epothilone A. Surprisingly, the desaturation of the C(10)?C(11) bond in these analogs was associated with a virtually complete loss in antiproliferative activity, which likely reflects a requirement for a ca. 60 ° C(10)?C(11) torsion angle in the tubulin‐bound conformation of 12,13‐trans‐epothilones.     

Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues.     

Stereospecific synthesis of 11S, 12S-oxido 5Z, 7E, 9E, 14Z-eicosatetraenoic acid

The first stereospecific synthesis of 11S,12S-oxido 5Z, 7E, 9E, 14Z-eicosatetraenoic acid has been achieved from 2-deoxy-D-ribose using either a Horner-Emmons or Wittig condensation to form the 9,10-trans or the 5,6-cis-double bond respectively.     

Total synthesis and antitumor activity of 12,13-desoxyepothilone F: an unexpected solvolysis problem at C15, mediated by remote substitution at C21

A new epothilone analogue, 12,13-desoxyepothilone F (dEpoF, 21-hydroxy-12,13-desoxyepothilone B, 21-hydroxyepothilone D), was synthesized and evaluated for antitumor potential. A convergent strategy employed for the semipractical synthesis of 12,13-desoxyepothilone B (dEpoB) has been utilized to yield an amount of dEpoF sufficient for relevant biological studies. The results from an in vitro assay reveal that this new analogue is highly active against various tumor cell lines with a potency comparable to that of dEpoB. In particular, the growth of resistant tumor cells is inhibited by dEpoF at concentrations where paclitaxel (Taxol) is basically ineffective. A preliminary assessment of its in vivo activity is also promising. The new analogue, containing an additional hydroxyl group at C21, exhibits advantages over other epothilones in terms of water solubility, and can serve as a readily functionalizable handle to produce other useful compounds for pertinent biological studies.     

Mechanism and stereospecificity of a fully saturating polyketide synthase module: nanchangmycin synthase module 2 and its dehydratase domain

Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxypyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed k(cat) 0.5 ± 0.1 min(-1) and K(m)(1) 19 ± 5 mM at 0.5 mM MM-CoA and k(cat)(app) 0.26 ± 0.02 min(-1) and K(m)(MM-CoA) 0.11 ± 0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(-)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2.     

Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases

Background: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thloester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino aryl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA.Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His₆, fusion protein, and purified in a single step by affinity chromatography. Incorporation of β-[³H]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4′-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro.Conclusions: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthees, and can also perform sequential steps in trans, as do multienzyme complexes.     

Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase

Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).     

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.     

Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase

BACKGROUND: Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest. RESULTS: The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains'). CONCLUSIONS: The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics.