Structure and mechanism of assembly line polyketide synthases

Robbins, Thomas; Liu, Yu Chen; Cane, David E.; Khosla, Chaitan

doi:10.1016/j.sbi.2016.05.009

Cited by 120 publications

(140 citation statements)

References 48 publications

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“…Earlier studies have highlighted a role for ACP-KS interactions in the channeling of polyketide chains between modules (13, 18); this conclusion is reinforced by our observation that a Glu 3 Lys charge reversal mutation in the ACP domain of the donor module DEBS M1 improved turnover in the presence of DEBS M3ϩTE. The structural logic of these protein-protein interactions is starting to become apparent (24). We note that, because interactions between the donor ACP and acceptor KS domains have not been explicitly interrogated in the experiments summarized in Table 1, the possibility of additional unproductive and therefore catalytically silent binding between chimeric PKS modules cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Klaus

Ostrowski

Austerjost

et al. 2016

Journal of Biological Chemistry

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The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

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Section: Discussionmentioning

confidence: 99%

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Klaus

Ostrowski

Austerjost

et al. 2016

Journal of Biological Chemistry

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“…Mutagenesis-guided docking studies 17–19 suggest that S315 interacts with the Ppant arms of the ACP domains involved in both chain translocation and chain elongation. By destabilizing both KS-ACP complexes, the S315A mutation could reduce the rate of either reaction.…”

Section: Discussionmentioning

confidence: 99%

Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase

et al. 2016

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Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C–C bond formation. S315 played a role in coupling decarboxylation to C–C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.

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“…1,2 The basic unit of the module consists of a β -ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP). The KS domain receives the growing polyketide chain from the upstream module and subsequently catalyzes chain elongation with an ACP-bound extender unit as the co-substrate (Figure 1).…”

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confidence: 99%

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

Mathews

Allison

Robbins³

et al. 2017

Biochemistry

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The crystal structure of the trans-acyltrans-ferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed.

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Structure and mechanism of assembly line polyketide synthases

Cited by 120 publications

References 48 publications

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

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