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Molecular Determinants of Natural Product Heterocyclization

天然产物杂环化的分子决定因素

基本信息

批准号：
9304455
负责人：
Daniel P. Dowling
金额：
$ 44.54万
依托单位：
UNIVERSITY OF MASSACHUSETTS BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9304455
关键词：
Active Sites Adopted Amino Acids Anabolism Antibiotics Antifungal Agents Antineoplastic Agents Antiviral Agents Area Bacitracin Binding Binding Sites Biochemical Biochemistry Biological Biological Assay Biomedical Engineering Bleomycin Breathing Catalysis Chemical Structure Chemicals Coin Crystallization Cysteine Data Development Drug Design Educational process of instructing Engineering Enzymes Epothilones Exposure to FDA approved Family Fluorescence Future Gene Cluster Generations Genomics Goals Hybrids Hydrolysis Institution Kinetics Libraries Link Mass Spectrum Analysis Methods Mission Molecular Molecular Biology Molecular Conformation Mutagenesis Mutation Natural Products Nature Pathway interactions Peptides Physical condensation Play Production Protein Engineering Proteins Reaction Research Resistance Resolution Roentgen Rays Role Route Sampling Series Serine Shapes Site Source Specificity Structure Substrate Specificity System Testing Therapeutic Threonine Time Translating Work X-Ray Crystallography anticancer activity assay development base biological systems biosynthetic product chemical function chemical kinetics design design and construction drug candidate drug development drug discovery enzyme activity graduate student inhibitor/antagonist interest microbial mutant novel peptide synthase preference prevent programs protein folding protein protein interaction screening structural biology therapeutic enzyme undergraduate student yersiniabactin

项目摘要

The overarching theme of the proposed research plan is to advance our ability to engineer natural product biosynthetic pathways for the production of candidate drug-like molecules. Natural products produced in Nature display a broad range of chemical functionalities, shapes, and sizes, imbuing many of these molecules with important therapeutic benefits that include anticancer and antibiotic activities. By increasing our understanding of these biosynthetic pathways at the atomic level, we will be able to manipulate different natural product systems to produce target compounds of chemical interest for drug discovery. This proposal aims to characterize the molecular and biochemical features of five-membered heterocycle formation in nonribosomal peptide synthetases (NRPSs), a family of natural products responsible for the generation of peptides and peptide/polyketide hybrid molecules such as the anticancer agents bleomycin and the epothilones, as well as the antibiotic bacitracin. The NRPS heterocyclization (HC or Cy) domain is responsible for installing five-membered thiazoline or oxazoline rings within NRPS products, originating from the amino acids cysteine or serine/threonine, respectively. The presence of these heterocycles increases compound stability and modifies the 3D shape of the natural products, therefore they play extremely important roles in defining biological activity and the ability to engineer these rings into novel compounds is of utmost interest. Aim 1 entails crystallizing different designed HC domain constructs in order to obtain a series of X-ray crystal structures of different HC domains from the epothilone and yersiniabactin biosynthetic gene clusters. These results will provide a direct comparison of how different HC domains from a related system are modified to accommodate differently sized substrate molecules. The second aim of this proposal focuses on rationally modifying substrate-binding residues within the HC domain and characterizing the resulting changes on substrate preferences and reaction kinetics. This aim will translate information regarding these structures to engineering applications, an important step to guide engineering of this system for future drug design. The overall strength of this AREA proposal is in the integrated approach at defining the activity of the NRPS HC domain for bioengineering efforts. This work combines the use of protein construct design, X-ray crystallography, mutagenesis, and biochemical activity assays to characterize the molecular function of the HC domain. In addition to directly involving undergraduate and graduate students in research, these results will impact multiple fields that include natural product biosynthesis, bioengineering, protein design and biocatalysis.

提出的研究计划的首要主题是提高我们设计天然产品的能力