Holzbaur I E, Harris R C, Bycroft M, Cortes J, Bisang C, Staunton J, Rudd B A, Leadlay P F
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Chem Biol. 1999 Apr;6(4):189-95. doi: 10.1016/S1074-5521(99)80035-0.
Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules.
The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer.
In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.
聚酮化合物是具有重要医学活性的化合物。聚酮合酶（PKS）多酶的模块化性质引发了人们对生物工程新型PKS的兴趣。然而，新型PKS的合理设计需要更深入了解天然PKS模块中起作用的立体控制机制。
将天然β-酮二酮中间体的N-乙酰半胱胺（NAC）硫酯衍生物与DEBS1-TE一起孵育，DEBS1-TE是红霉素PKS的一种衍生物，仅包含模块1和2。DEBS1-TE的两个酮还原酶（KR）结构域的还原产物是相应β-羟基二酮NAC硫酯的（2S，3R）和（2R，3S）异构体的混合物。使用仅包含KR1的DEBS1-TE突变体重复孵育，仅产生（2S，3R）异构体。
与早期结果相反，KR1仅选择（2S）异构体并将其立体定向还原为（2S，3R）-3-羟基-2-甲基酰基产物。模块1的KR结构域控制羟基二酮中间体中含甲基和羟基的手性中心的立体化学结果。然而，早期工作表明模块2中产生并与酶结合的正常酮酯不会发生差向异构化。因此，C-2处的立体化学是通过仅生成（2R）-酮酯的缩合反应建立的，而C-3处的立体化学是通过酮基的还原建立的。因此，红霉素PKS的模块1和2中存在两种不同的立体化学控制机制。这些结果应为设计杂合PKS以在聚酮化合物产物中产生改变的立体化学提供更合理的基础。
