Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases
Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases
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DOI：
10.1021/bi012086u
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发表时间：
2002-04-16
期刊：
影响因子：
2.9
通讯作者：
Khosla, C
中科院分区：
文献类型：
--
作者：
Wu, N;Cane, DE;Khosla, C
6-脱氧赤酮内酯 B 合酶 (DEBS) 是模块化聚酮合酶 (PKS)，负责 6-dEB 的生物合成，6-dEB 是抗生素红霉素的苷元核心。 6-dEB 的生物合成通过 DEBS 的六个模块以装配线方式进行，每个模块催化一组专用反应，因此最终产品的结构由模块沿装配线的排列决定。蛋白质序列和酶功能之间的这种透明关系对于所有模块化 PKS 来说都是常见的，并使这些酶成为通过模块交换进行蛋白质工程的有吸引力的支架。与模块交换相关的仍需要解决的基本问题之一是将中间体从一个模块引导到下一个模块的机制。虽然先前已表明相邻多肽 N 端和 C 端的短接头区域在介导模块间转移中发挥重要作用，但尚未探讨其他蛋白质-蛋白质相互作用的贡献。在这里，我们研究了接头相互作用的作用以及供体酰基载体蛋白（ACP）结构域和下游酮合酶（KS）结构域在各种情况下的相互作用。链接器相互作用和ACP-KS相互作用在DEBS中的模块2-模块3和模块4-模块5接口处做出相对相等的贡献。相反，模块 2 和 6 对非天然 ACP 结构域呈现的底物更具耐受性。这种耐受性被用于工程混合 PKS-PKS 和 PKS-NRPS（非核糖体肽合成酶）连接，并为未来工程新型嵌合 PKS 提出了基本规则。
6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB, the aglycon core of the antibiotic erythromycin. The biosynthesis of 6-dEB proceeds in an assembly-line fashion through the six modules of DEBS, each of which catalyzes a dedicated set of reactions, such that the structure of the final product is determined by the arrangement of modules along the assembly line. This transparent relationship between protein sequence and enzyme function is common to all modular PKSs and makes these enzymes an attractive scaffold for protein engineering through module swapping. One of the fundamental issues relating to module swapping that still needs to be addressed is the mechanism by which intermediates are channeled from one module to the next. While it has been previously shown that short linker regions at the N- and C-termini of adjacent polypeptides play an important role in mediating intermodular transfer, the contributions of other protein-protein interactions have not yet been probed. Here, we investigate the roles of the linker interactions as well as the interactions between the donor acyl carrier protein (ACP) domain and the downstream ketosynthase (KS) domain in various contexts. Linker interactions and ACP-KS interactions make relatively equal contributions at the module 2-module 3 and the module 4-module 5 interfaces in DEBS. In contrast, modules 2 and 6 are more tolerant toward substrates presented by nonnatural ACP domains. This tolerance was exploited for engineering hybrid PKS-PKS and PKS-NRPS (nonribosomal peptide synthetase) junctions and suggests fundamental ground rules for engineering novel chimeric PKSs in the future.