Böhm I, Holzbaur I E, Hanefeld U, Cortés J, Staunton J, Leadlay P F
Cambridge Centre for Molecular Recognition, University of Cambridge, UK.
Chem Biol. 1998 Aug;5(8):407-12. doi: 10.1016/s1074-5521(98)90157-0.
Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compounds. Polyketides are biosynthesised in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochemical outcome of polyketide chain extension using genetic engineering of domains within each module.
We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1-2) consists of a single chimaeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1-2 was expressed in S. erythraea, the strain preferentially6 accumulated the diketide (2R, 3S)-2-methyl-3-hydroxy pentanoic acid.
These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2methyl-3-hydroxy intermediate by epimerising the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The observed formation of the alternative (2R)-methyl-3-hydroxy product catalysed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.
聚酮化合物是一类庞大且结构多样的天然产物，包括抗生素、抗真菌剂和免疫抑制化合物。聚酮化合物在丝状细菌中由模块化聚酮合酶（PKS）生物合成，其中每个链延伸循环都需要不同的酶活性“模块”。最近提出的模块化PKS二聚体模型预测，即使是单模块PKS在没有其他PKS组件的情况下也应具有催化活性。研究人员还对通过对每个模块内的结构域进行基因工程操作来控制聚酮链延伸的立体化学结果感兴趣。
我们从糖多孢红霉菌产生红霉素的PKS（DEBS）构建了一个最小模块化PKS。二酮合酶（DKS1-2）由单个嵌合延伸模块组成，该模块源自DEBS模块1酮酰基-ACP合酶（KS），夹在加载模块和链终止硫酯酶之间。当DKS1-2在糖多孢红霉菌中表达时，该菌株优先积累二酮（2R，3S）-2-甲基-3-羟基戊酸。
这些结果表明，如预测的那样，即使是单模块PKS在没有其他DEBS蛋白的情况下也具有催化活性。在其正常环境中，酮合酶结构域KS1被认为通过将碳-碳链形成的初始产物（2R）-2-甲基-3-酮酯差向异构化来生成（2S）-2-甲基-3-羟基中间体。由DKS1-2催化观察到的替代（2R）-甲基-3-羟基产物的形成为此提议提供了有力支持，并表明如何在模块化PKS上实现立体特异性的靶向改变。
