Assembly line polyketide synthases: mechanistic insights and unsolved problems

Abstract

Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.

Figure 1

Assembly line organization of the 6-deoxyerythronolide B synthase (DEBS). (A) DEBS is an ∼2 MDa α₂β₂γ₂ protein assembly that harbors six elongation modules (modules 1–6) flanked by a loading didomain (LD) and a thioesterase (TE). It catalyzes the conversion of 1 equiv of propionyl-CoA and 6 equiv of (2S)-methylmalonyl-CoA into 6-deoxyerythronolide B, using 6 equiv of NADPH as a cofactor. Each module harbors the necessary enzymatic activity for one round of chain elongation and associated modifications of the growing polyketide chain. The reaction intermediates shown attached to the ACP domain of each module correspond to the final products of each of the respective modules. (B) Module 3 is a representative catalytic module within the DEBS assembly line. Its active sites are shown, as is the overall transformation catalyzed by this set of active sites. ACP is the acyl carrier protein, AT acyltransferase, KS ketosynthase, and KR⁰ a ketoreductase homologue that lacks NADPH-dependent reductase activity but retains epimerase activity.

Figure 2

Ribbon diagram representations of atomic structures of prototypical domains and didomains from assembly line polyketide synthases. In figures showing KR and ER domains, the bound NADPH cofactor is also shown. All structures were derived from components of DEBS itself, with the exception of the ER-KR didomain obtained from the spinosyn synthase. For details, see refs (−9).

Figure 3

Individual reactions in the catalytic cycle of DEBS module 3. Each module of DEBS catalyzes a set of reactions that can be categorized as follows: (1) intermodular chain translocation involving transthioesterification from the ACP domain of the upstream module to the KS domain of the target module, (2) transfer of an acyl group from an α-carboxyacyl-CoA extender unit to the ACP domain of the target module, (3) chain elongation involving decarboxylative condensation of the growing polyketide chain onto the extender unit, (4) modification of the newly elongated chain at the α- and β-carbon atoms, and (5) intermodular chain translocation involving transthioesterification from the ACP domain of the target module to the KS domain of the downstream module. In the case of the representative DEBS module 3 shown here, chain modification simply involves epimerization of the α-carbon, a reaction that is catalyzed by KR⁰. Note that the three proposed states of the ketosynthase are highlighted as KS, KS*, and KS**. For details, see the text.

Figure 4

Transformations catalyzed by module 4 of DEBS. Postelongation chain modification reactions include (3) ketoreduction, (4) dehydration, and (5) enoyl reduction.

Figure 5

Key reactions catalyzed by the first three modules of the hybrid assembly line responsible for epothilone biosynthesis. The first three modules of this synthetase comprise a polyketide synthase (PKS) module (light green), followed by a nonribosomal peptide synthetase (NRPS) module (yellow), followed by another PKS module (dark green). The first module harbors a KS⁰ domain that catalyzes the decarboxylation of malonyl-ACP, yielding acetyl-ACP. (This reaction is not explicitly shown.) The condensation (C) domain of the second module then catalyzes condensation between acetyl-ACP on module 1 and cysteinyl-PCP on module 2; this reaction is accompanied by concomitant translocation of the growing chain from the PKS to the NRPS module. The NRPS module also catalyzes chain modification via cyclization (Cy) and oxidation (Ox), yielding a PCP-bound thiazole moiety. This intermediate then undergoes translocation onto the KS domain of the downstream PKS module in a manner that is entirely analogous to the downstream translocation event shown in Figure 3.