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

研究期限

未知 / 未知

项目级别

国家级

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.