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Molecular Analysis of Modular Polyketide Synthases

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

BOND, MICHELLE RUEFFER

项目受资助机构

UNIVERSITY OF MICHIGAN AT ANN ARBOR

项目编号

5R01GM076477-12

立项年度

2018

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

388963.00美元

学科

Bioengineering

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

SHERMAN, DAVID H ; SMITH, JANET L.

参与机构

NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

项目标书摘要：DESCRIPTION (provided by applicant): Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This renewal application of a highly productive collaborative program proposes to employ the versatile and well-characterized Streptomyces Venezuela pikromycin PKS, as well as the erythromycin, tylosin, curacin and bryostatin pathways which were the subjects of expanded detailed analysis during the previous cycle of support and are now poised for major new progress. These systems each bear fascinating biochemical features that will expand our understanding of the specificity and structural characteristics that lead to biological activity within and between natie and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, keto group processing, and termination that lea to the remarkable chemical diversity of polyketide natural products. Detailed biochemical analysis, along with X-ray and cryoEM structural biology, and molecular dynamics approaches will be applied to probe substrate specificity. Moreover, synthetic chemistry of natural and near-natural substrates will be employed to develop chemoenzymatic approaches to enable pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents. Specific aims include: I. Molecular analysis of bacterial modular polyketide synthases. We will design and employ natural and unnatural synthetic substrates and extender units to explore selectivity and tolerance in chain loading, elongation and processing in the terminal modules of Pik (modules 5 and 6), DEBS (modules 5 and 6), Tyl (modules 6 and 7), and select Cur PKS modules. II. Develop mutational strategies to engineer modular PKSs with greater catalytic efficiency toward unnatural substrates. A high-throughput bioactivity-based screen will be developed to assess the efficiency of mutant PKS modules for improved activity toward target unnatural substrates. III. Molecular analysis of bacterial symbiont trans-AT modular PKSs and ?ranching. We will explore the protein recognition determinants for trans-AT interactions, substrate selectivity, and structure and function using synthetic substrates, biochemical analysis, x-ray crystallography, cryoEM, and FT-ICR MS. In addition, a proof-of-concept method will be developed to interrogate biochemical function using bryostatin (Bry) PKS modules 3 and 4 and BryP/surrogate trans-ATs and ?ranching enzymes.

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  • 1.A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

    • 关键词：
    • ERYTHROMYCIN POLYKETIDE SYNTHASE; NONRIBOSOMAL PEPTIDE SYNTHETASE;SUBSTRATE-SPECIFICITY; ACYLTRANSFERASE DOMAIN; FUNCTION MUTAGENESIS;BIOSYNTHESIS; MACROLACTONIZATION; MECHANISM; LACTONE;10-DEOXYMETHYNOLIDE
    • Koch, Aaron A.;Hansen, Douglas A.;Shende, Vikram V.;Furan, Lawrence R.;Houk, K. N.;Jimenez-Oses, Gonzalo;Sherman, David H.
    • 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》
    • 2017年
    • 139卷
    • 38期
    • 期刊

    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 rnacrolactones. 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.

    ...

  • 3.Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization

    • 关键词：
    • PRECURSOR-DIRECTED BIOSYNTHESIS; SYNTHASE DOCKING DOMAINS;MICROMONOSPORA-ROSARIA; GLYCOSIDATION REACTION; O-METHYLTRANSFERASES;TYLOSIN; PIKROMYCIN; MYCINAMICIN; ROSAMICIN; ALCOHOLS
    • Lowell, Andrew N.;DeMars, Matthew D., II;Slocum, Samuel T.;Yu, Fengan;Anand, Krithika;Chemler, Joseph A.;Korakavi, Nisha;Priessnitz, Jennifer K.;Park, Sung Ryeol;Koch, Aaron A.;Schultz, Pamela J.;Sherman, David H.
    • 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》
    • 2017年
    • 139卷
    • 23期
    • 期刊

    Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

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