Ranganathan A, Timoney M, Bycroft M, Cortés J, Thomas I P, Wilkinson B, Kellenberger L, Hanefeld U, Galloway I S, Staunton J, Leadlay P F
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK.
Chem Biol. 1999 Oct;6(10):731-41. doi: 10.1016/s1074-5521(00)80020-4.
Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function.
We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules.
Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.
聚酮化合物是结构多样的天然产物，具有一系列医学上有用的活性。非芳香族细菌聚酮化合物是在模块化聚酮合酶（PKS）多酶上合成的，其中每个链延伸循环都需要不同的酶活性“模块”。尝试设计和构建合成特定新型聚酮化合物的模块化PKS，对我们对PKS结构和功能的理解提供了特别严格的测试。
我们基于DEBS1-TE构建了双模块和三模块PKS，DEBS1-TE是红霉素PKS的衍生物，仅包含模块1和2以及硫酯酶（TE），通过用源自雷帕霉素PKS的合适对应物替换多个结构域。获得了合成预测目标三酮内酯的杂合PKS，三酮内酯是降胆固醇他汀类药物的简单类似物。在构建模块间融合时，无论是在同一蛋白质还是不同蛋白质中的模块之间，发现保留跨越连续模块之间连接点的完整酰基载体蛋白-酮合成酶（ACP-KS）双结构域是有利的。
相对简单的考虑因素决定了功能性杂合PKS的构建。融合位点应选择在酶结构域之间表面可及的连接区域，如先前所示，或就在结构域保守边缘的内侧。ACP结构域与相邻KS结构域的相互作用，无论是否在同一聚酮化合物上，都特别重要，这既通过保留适当的蛋白质-蛋白质相互作用，也通过优化酰基链从一个模块的ACP到下游模块的KS的关键转移中改变的聚酮化合物链的分子识别。
