View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Polyketide synthesis in vitro on a modular polyketide synthase Kirsten EH Wiesmannl, Jesus Cortkl, Murray JB Brown*, Annabel 1 Cutter*, James Staunton*, and Peter F Leadlay’* ‘Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, Cambridge CB2 IQW, UK, Xambridge Centre for Molecular Recognition and Department of Organic Chemistry, University of Cambridge, Cambridge CB2 1 EW, UK Background: The 6-deoxyerythronolide B synthase out to purify the chimaeric enzyme and to determine its (DEBS) of Sacckavopolyspora erytkraea, which synthesizes activity in vitro. the aglycone core of the antibiotic erythromycin A, con- Results: The purified DEBSl-TE multienzyme catalyzes tains some 30 active sites distributed between three mul- synthesis of triketide lactones in vitro.The synthase specif- tienzyme polypeptides (designated DEBSl-3). This ically uses the (25’)-isomer of methylmalonyl-CoA, as complexity has hitherto frustrated mechanistic analysis of previously proposed, but has a more relaxed specificity for such enzymes. We previously produced a mutant strain of the starter unit than in vivo. S. erytkraea in which the chain-terminating cyclase Conclusions: We have obtained a purified polyketide domain (TE) is fused to the carboxyl-terminus of synthase system, derived from DEBS, which retains cat- DEBSl, the multienzyme that catalyzes the first two alytic activity. This approach opens the way for mechanis- rounds of polyketide chain extension in S. erytkraea. This tic and structural analyses of active multienzymes derived mutant strain produces triketide lactone in vivo. We set from any modular polyketide synthase. Chemistry & Biology September 1995, 2:583-589 Key words: cyclase, erythromycin, polyketide synthase, Saccharopolyspora erythraea, triketide lactone Introduction housed in the three multienzyme polypeptides [8] of Complex polyketides are natural products, found pre- 6-deoxyerythronolide B synthase (DEBS) (Fig. 1). The dominantly in Streptomyces and related filamentous bacte- most remarkable example studied thus far is the PKS for ria, that exhibit an impressive range of antibiotic, the immunosuppressant rapamycin from Stveptomycer anticancer, antiparasite and immunosuppressant activities. hygrorcopicus, which contains 14 modules contained Despite their apparent structural diversity, they are syn- within only three huge multienzyme polypeptides [9]. thesi.zed by a common pathway in which units derived from acetate or propionate are condensed onto the Progress in understanding the enzymology of such growing chain in a process resembling fatty acid biosyn- modular type I systems has been frustrated by the lack of thesis [1,2]. The intermediates remain bound to the a cell-free system to study polyketide chain synthesis by polyketide synthase (PKS) during multiple cycles of any of these multienzymes, although several partial reac- chain extension and (to a variable extent) reduction of tions of DEBS have been successfully assayed in vitro the B-keto group formed in each condensation. The [7,10,11]. We recently introduced [12] a novel, efficient structural variation between naturally occurring poly- and potentially general method for the controlled early ketides arises largely from the way in which each PKS release of polyketide chains from a modular type I PKS, controls the number and type of units added, and from by relocation of the cyclase/thioesterase (TE) domain the extent and stereochemistry of reduction at each from DEBS3, which normally catalyzes the fifth and cycle. Still greater diversity is produced by the action of sixth rounds of chain extension, to the carboxyl-terminus regiospecific glycosylases, methyltransferases and oxidative of DEBSl, which catalyzes the first two rounds (Fig. 1). enzymes on the product of the PKS. Mutants of S. erythruea [12] or of S. coelicolor [13] that lack endogenous DEBS genes but express the chimaeric Complex reduced polyketides, such as the macrolides DEBSl-TE fusion protein were shown to produce the and polyethers, are synthesized in bacteria on multifunc- expected triketide lactone 1B (Fig. 1) in good yield, tional or type I PKSs that contain a separate set, or together with variable amounts of a second triketide module, of enzymatic activities for every round of chain lactone 1A. extension 13-61. For example, the clinically-important antibiotic erythromycin A is derived from propionyl- We show here that cell extracts from such a strain are CoA and six molecules of (2S)-methylmalonyl-CoA [7], also able to synthesize triketide lactones 1A and lB, through the sequential action of six such modules in the presence of methylmalonyl-CoA, an acyl-CoA *Corresponding author. 0 Current Biology Ltd ISSN 1074-5521 583 584 Chemistry & Biology 1995, Vol 2 No 9 Fig. 1. The domain organization of the ‘triketide lactone synthase’ (DEBSl-TE). Each protein module contains an acyl carrier protein (ACP), an acyltransferase (AT), a B-ketoacyl-ACP synthase (KS) and B-ketoacyl-ACP reductase (KR). The thioesterase-cyclase (TE) releases the triketide lactone product (normally compound 1 B) from the synthase [I 21. HO R lA:R=Me 1 B: R = Et 1C: R = n-Pr 1D: R= i-Pr (such as propionyl-CoA), and NADPH. Furthermore, either by thin-layer chromatography (TLC), or by DEBSl-TE has been purified from the extracts to normal-phase or reverse-phase high-pressure liquid homogeneity and retains its activity. We report here the chromatography (HPLC), and their relative mobilities or use of this in vitro system to explore several important elution times were compared with those of authentic aspects of the specificity and stereospecificity of a typical [14C]-triketide lactone 1B. modular polyketide synthase. A typical profile for the production of radiolabelled com- Results and discussion pound 1B over time is shown in Fig. 2, in an experiment Demonstration of cell-free polyketide biosynthesis in which the extract was incubated with propionyl-CoA, Cell extracts were prepared from the DEBSl-TE- (R,S)-methylmalonyl-CoA, and NADPH. Purified expressing S. evythraea strain JCBlOl as described in methylmalonyl-CoA epimerase from Propionibactevium Materials and methods, and used immediately in experi- shermanii [14] was added to ensure rapid equilibration of ments to detect the conversion of propionyl-CoA and the two stereoisomers, as only (2S)-methylmalonyl-CoA methylmalonyl-Cob into triketide lactone 1B. Radio- is apparently used by DEBS [7]. activity was added in the form of either [14C]-propionyl- CoA or [14C]-(R,S)-methylmalonyl-CoA, or both, and Although the concentrations of propionyl-CoA and the reaction mixtures were quenched by extraction with methylmalonyl-Cod used here are unlikely to be ethyl acetate. The radioactive products were separated optimal, this simple in vitro system has allowed several Fig. 2. Cell extracts containing DEBSI-TE generate triketide lactones in vitro. The figure shows production of 14C-labelled triketide lactones IA and IB from 14C-labelled 300 t.t.M propi- onyl-CoA, 600 ~.LM (27nCi) (RS)-methyl- malonyl-CoA, and 600 FM NADPH in Time (mins) the presence of methylmalonyl-CoA epimerase in cell extracts of S. erytbraea at different time points. The lactones were separated on silica TLC plates and radioactivity was detected by two- dimensional phosphorimaging. Lactone 1A has R, 0.58, 1 B has R, 0.47. The inset summarizes the increase in production of IA (lower curve) and 1B (upper curve) with time. Production is mea- sured in arbitrary units. The material at Rf 0.28 is unidentified. For details see the text. Polyketide synthesis in vitro Wiesmann et al. 585 other features of polyketide chain extension to be exam- DEBSl-thioesterase has been shown previously to ined, for example, by using well known inhibitors of spe- produce a mixture of compounds 1A and lB, with com- cific enzymatic activities.We previously showed [14] that pound 1A as the major product [13]. For reasons that are the active site serine of DEBSl-TE, Ser3029, reacts not clear, DEBSl alone, when expressed using the iden- rapidly with the serine protease inhibitor phenyl- tical system in S. coelicolor, is reported by others to methanesulfonyl fluoride (PMSF), and that alteration of produce only compound 1B [17]. this residue by site-directed mutagenesis caused a > lOO- fold decrease in the rate of triketide lactone synthesis by Polyketide production by purified DEBSl-TE chimaeric DEBSl-TE in vivo [12]. We therefore antici- The DEBSl-TE multienzyme was purified to 90-95 % pated that PMSF would affect chain release in the in vitro homogeneity from S. erythmeu JCBlOl by methods that system, and indeed addition of 1mM PMSF reduced were previously successful for the native DEBS multien- produ.ction of the radiolabelled compound 1B to 65% of zymes [8,10] and for DEBSS over-expressed in Esckerickiu the amount produced in the control incubation (data not coli [18] .The purified enzyme was used in another series shown). Cerulenin is an inhibitor of fatty acid synthases of experiments to establish the extent to which alterna- from almost all sources [15], and also inhibits production tive starter units could be used by the polyketide synthase. of compound 1B. Compared to a control incubation, the The incubations contained [14C]-methylmalonyl-CoA presence of 0.1 mM cerulenin reduced the rate of incor- and either acetyl-, propionyl-, n-butyryl-, iso-butyryl-, poration 2-fold, and