Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases
Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases
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DOI：
10.1074/jbc.m116.730531
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发表时间：
2016-07-29
影响因子：
4.8
通讯作者：
Khosla, Chaitan
中科院分区：
文献类型：
--
作者：
Klaus, Maja;Ostrowski, Matthew P.;Khosla, Chaitan
重组完整的聚酮合成酶(PKS)模块的可能性已被广泛探索。酶-底物相互作用和蛋白质-蛋白质相互作用都会影响嵌合蛋白激酶的活性，但它们的相对贡献尚不清楚。我们现在通过研究一个包含红霉素、利福霉素和雷帕霉素合成酶模块的11个双模块和8个三模块嵌合PKS的文库来解决这个问题。尽管许多嵌合体产生了可检测到的产物，但几乎所有嵌合体的比活性都低于参考天然PKS的10%。对选定的具有相同上游模块的双模嵌合体的分析表明，周转率与模块间链转位的效率有关。在一个嵌合体中，上游模块的酰基载体蛋白(ACP)结构域在一个残基上发生突变，预计会影响酮合成酶-ACP的识别，从而提高周转率。相反，用产生对映体ACP结合的二酮的平行对数取代上游模块的酮还原酶结构域，与天然二酮相比，六个异源下游模块的处理速率没有变化。综上所述，这些结果表明，在催化高效嵌合PKS的进化或设计中，蛋白质-蛋白质相互作用比酶-底物识别发挥更大的作用。
The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.