81644-19-1Relevant academic research and scientific papers
A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst
Koch, Aaron A.,Hansen, Douglas A.,Shende, Vikram V.,Furan, Lawrence R.,Houk,Jiménez-Osés, Gonzalo,Sherman, David H.
, p. 13456 - 13465 (2017)
Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.
Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides
Hansen, Douglas A.,Koch, Aaron A.,Sherman, David H.
, p. 13450 - 13455 (2017)
Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate-limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified pikromycin (Pik) pentaketides that mimic early pathway engineering to probe the substrate tolerance of the PikAIII-TE module in vitro. Truncated pentaketides were processed with varying efficiencies to corresponding macrolactones, while pentaketides with epimerized chiral centers were poorly processed by PikAIII-TE and failed to generate 12-membered ring products. Isolation and identification of extended but prematurely offloaded shunt products suggested that the Pik thioesterase (TE) domain has limited substrate flexibility and functions as a gatekeeper in the processing of unnatural substrates. Synthesis of an analogous hexaketide with an epimerized nucleophilic hydroxyl group allowed for direct evaluation of the substrate stereoselectivity of the excised TE domain. The epimerized hexaketide failed to undergo cyclization and was exclusively hydrolyzed, confirming the TE domain as a key catalytic bottleneck. In an accompanying paper, we engineer the standalone Pik thioesterase to yield a thioesterase (TES148C) and module (PikAIII-TES148C) that display gain-of-function processing of substrates with inverted hydroxyl groups.
Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units
Kalkreuter, Edward,Crowetipton, Jared M.,Lowell, Andrew N.,Sherman, David H.,Williams, Gavin J.
supporting information, p. 1961 - 1969 (2019/02/05)
There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field.
Substrate controlled divergence in polyketide synthase catalysis
Hansen, Douglas A.,Koch, Aaron A.,Sherman, David H.
supporting information, p. 3735 - 3738 (2015/04/14)
Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of PikAIV, the sixth and final module of the pikromycin (Pik) pathway. We demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. This outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in PKS functional studies and biocatalysis.
Biocatalytic synthesis of pikromycin, methymycin, neomethymycin, novamethymycin, and ketomethymycin
Hansen, Douglas A.,Rath, Christopher M.,Eisman, Eli B.,Narayan, Alison R. H.,Kittendorf, Jeffrey D.,Mortison, Jonathan D.,Yoon, Yeo Joon,Sherman, David H.
, p. 11232 - 11238 (2013/08/23)
A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of d-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.
Frontiers and opportunities in chemoenzymatic synthesis
Mortison, Jonathan D.,Sherman, David H.
scheme or table, p. 7041 - 7051 (2010/12/20)
Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.
Total synthesis of 10-deoxymethynolide and narbonolide
Xuan, Richeng,Oh, Hong-Se,Lee, Younghoon,Kang, Han-Young
, p. 1456 - 1461 (2008/09/16)
(Chemical Equation Presented) A flexible and convenient approach was developed for the synthesis of 10-deoxymethynolide (1) and narbonolide (2), which are aglycones of the methymycin and the pikromycin families of macrolide antibiotics. These lactones are
Macrolactonization to 10-deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin/methymycin polyketide synthase
He, Weiguo,Wu, Jiaquan,Khosla, Chaitan,Cane, David E.
, p. 391 - 394 (2007/10/03)
The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast
Chain elongation, macrolactonization, and hydrolysis of natural and reduced hexaketide substrates by the picromycin/methymycin polyketide synthase
Wu, Jiaquan,He, Weiguo,Khosla, Chaitan,Cane, David E.
, p. 7557 - 7560 (2007/10/03)
(Chemical Equation Presented) Dual action: An analogue of a natural hexaketide thioester substrate is incubated with recombinant picromycin/methymycin synthase (PICS) module 6 with its attached thioesterase (TE). A mixture of 12- and 14-membered-ring macrolactones 10-deoxymethynolide (1) and narbonolide (2) are generated (see scheme) by competing chain elongation and direct lactonization of the substrate. KS = ketosynthase, AT = methylmalonyl transferase, ACP = acyl carrier protein, metmal CoA = methylmalonyl coenzyme A.
Biochemical investigation of pikromycin biosynthesis employing native penta- and hexaketide chain elongation intermediates
Aldrich, Courtney C.,Beck, Brian J.,Fecik, Robert A.,Sherman, David H.
, p. 8441 - 8452 (2007/10/03)
The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of chain e
