School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
Angew Chem Int Ed Engl. 2019 Sep 2;58(36):12446-12450. doi: 10.1002/anie.201905482. Epub 2019 Aug 7.
The presence of β-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential β-branching modifications. We use purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of β-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of C NMR of a single C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific β-branch.
具有生物活性的聚酮化合物结构中β-支链的存在，是这种结构特征广泛重要性的基础。卡里曼他汀是一种具有抗葡萄球菌选择性活性的聚酮类抗生素，其生物合成涉及三种不同的、连续的β-支链修饰的空前整合。我们使用纯化的卡里曼他汀生物合成机制的单域和多域酶成分，在体外解决卡里曼他汀中β-支链模式如何被控制的问题。对酶产物的有力鉴别需要开发一种通用的测定方法，该方法利用单个 C 标记物掺入关键生物合成模拟物的 C NMR 以及酰基辅酶 A 的有利动态特性。我们报告了一个以前未被分配的模块烯酰辅酶 A 水合酶（mECH）结构域，以及能够生成每个特定β-支链的酶构建体和级联的组装。
