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Radical SAM enzyme engineering to produce improved thiopeptide antibiotics

激进的 SAM 酶工程生产改进的硫肽抗生素

基本信息

批准号：
9065177
负责人：
Andrew Buller
金额：
$ 5.61万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2017-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The development and industrial production of antibiotics involves an intimate interplay between biological and synthetic chemistries. Biological systems have been selected throughout evolution to generate highly bioactive products through pathways with stereoselective catalytic activity and unmatched atom economy. However, natural products typically have poor pharmacological properties and synthetic chemistry is employed to modify them into useful drugs. These synthetic steps frequently feature low yields, involve the use of toxic reagents, and contribute substantially to the overall cost of drug development. Thus, the evolution of enzymes that circumvent these synthetic steps by operating on unnatural substrate analogues [and perform new biochemical transformations] is important for the efficient conversion of potent natural products into clinically-useful drugs. This proposal outlines a strategy to subvert the natural biosynthetic pathway of nosiheptide, a thiopeptide antibiotic, to generate nosiheptide analogues with improved activity and solubility. We have identified the radical SAM enzyme NosL, as an ideal target for this objective. Radical SAM enzymes perform chemically challenging reactions, such as the radical fragmentation-recombination reaction of NosL, in many important biosynthetic pathways. Directed evolution of radical SAM enzymes has not been performed and [we will use this opportunity to uncover efficient strategies for tuning their reactivity]. Our specific aims are: [(1) To expand the substrte profile of NosL through directed evolution; (2) Use NosL to establish efficient techniques for uncovering new RS chemistry and]; (3) Introduce engineered nosL genes into the native producer to ferment regiospecifically modified nosiheptides analogues. These antibiotics may then be further modified through cross-coupling chemistry to access pharmacologically-promising non-natural products. All precursors may be produced on preparative-scale with the promiscuous enzyme tryptophan synthase, synthesized with established techniques, or purchased from commercial vendors.

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Enantioselective, intermolecular benzylic C-H amination catalysed by an engineered iron-haem enzyme.

DOI：
10.1038/nchem.2783
发表时间：
2017-07
期刊：
Nature chemistry
影响因子：
21.8
作者：
Prier CK;Zhang RK;Buller AR;Brinkmann-Chen S;Arnold FH
通讯作者：
Arnold FH

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity.

DOI：
10.1021/acs.biochem.6b01127
发表时间：
2016-12-27
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Buller, Andrew R.;van Roye, Paul;Murciano-Calles, Javier;Arnold, Frances H.
通讯作者：
Arnold, Frances H.

A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation.

DOI：
10.1002/anie.201606242
发表时间：
2016-09-12
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
影响因子：
16.6
作者：
Murciano-Calles, Javier;Romney, David K.;Brinkmann-Chen, Sabine;Buller, Andrew R.;Arnold, Frances H.
通讯作者：
Arnold, Frances H.

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.