Structure-Based Dissociation of a Type I Polyketide Synthase Module

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Presentation transcript:

Structure-Based Dissociation of a Type I Polyketide Synthase Module Alice Y. Chen, David E. Cane, Chaitan Khosla Chemistry & Biology Volume 14, Issue 7, Pages 784-792 (July 2007) DOI: 10.1016/j.chembiol.2007.05.015 Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 1 DEBS Organization and Chemistry (A) Modular organization of 6-deoxyerythronolide B synthase (DEBS). Chain elongation occurs minimally through the combined action of the β-ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains. Optional tailoring domains including ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains control the ultimate oxidation state of the β carbon. Once processed, the polyketide chain is either passed to the KS domain of the downstream module or cyclized and released by the thioesterase (TE) domain at the C terminus of the polyketide synthase. LDD, loading didomain. (B) Chain elongation cycle catalyzed by DEBS module 3 + TE. AT is acylated with a methylmalonyl extender unit from its CoA derivative, which is then transferred to the phosphopantetheine arm of the ACP domain. KS is primed with a 2-methyl-3-hydroxy pentanoyl unit from diketide 1. Once both the growing chain and the extender units are covalently loaded onto the module, KS-mediated decarboxylative condensation occurs to form triketide β-ketoacyl-ACP intermediate 2 with the release of carbon dioxide. TE catalyzes the release of the final triketide ketolactone product 3. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 2 Fragmented Module Systems (A and B) Chain elongation performed by (A) [KS][AT] didomain and stand-alone ACP, and (B) stand-alone KS, AT, and ACP. (C) Reduction performed by stand-alone KR to produce product 4. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 3 Radio-SDS-PAGE for Protein Acylation (A) Acylation and transacylation activities of isolated DEBS AT3. Radiolabeled methylmalonyl-CoA with AT3(3) (lane 1), AT3(3) + ACP3 (lane 2), AT3(0) (lane 3), and AT3(0) + ACP3 (lane 4). (B) Acylation activities of isolated DEBS KS3. Radiolabeled diketide 1 with KS3 (lane 1), KS3 + AT3(3) (lane 2), and KS3 + AT3(0) (lane 3). Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 4 Effect of the Post-AT Linker in Condensation Radio-TLC/phosphorimaging assay for the formation of triketide ketolactone 3 by DEBS KS3 + AT3(3) + ACP3 (lane 1) and KS3 + AT3(0) + ACP3 (lane 2). This result indicates that the post-AT linker is required for KS-catalyzed β-ketoacyl-ACP synthase activity. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 5 Radio-TLC Assay to Examine the Specificity of KR toward the Partnering KS DEBS KR1 (lane 1), KR2 (lane 2), and KR6 (lane 3) with [KS3][AT3] + ACP3 (A) or [KS6][AT6] + ACP6 (B). In both module 3 and module 6 systems, KR1 produced no reduced product, KR2 produced the reduced product exclusively, while KR6 produced a mixture of the reduced and unreduced products. Radioactive intensities of the products are tabulated in Table S1. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 6 Radio-TLC Assay to Examine the Specificity of KR toward the Partnering ACP DEBS ACP1–6 (lanes 1–6, respectively) with [KS3][AT3] + KR2 (A) or [KS6][AT6] + KR6 (B). Regardless of the ACP partner, KR2 produced the reduced product almost exclusively, while KR6 produced a mixture of the reduced and unreduced products. Radioactive intensities of the products are tabulated and compared to the kcat profiles in Table S2. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 7 Condensation Performed by the Fully Fragmented Systems Radio-TLC assay for the formation of triketide lactones 3 and 4 by the fully fragmented systems. DEBS KS3 + AT3(3) with no KR (lane 1), KR2 (lane 2), or KR6 (lane 3). Radioactive intensities of the products are tabulated in Table S3. Chemistry & Biology 2007 14, 784-792DOI: (10.1016/j.chembiol.2007.05.015) Copyright © 2007 Elsevier Ltd Terms and Conditions