Baerga-Ortiz Abel, Popovic Bojana, Siskos Alexandros P, O'Hare Helen M, Spiteller Dieter, Williams Mark G, Campillo Nuria, Spencer Jonathan B, Leadlay Peter F
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
Chem Biol. 2006 Mar;13(3):277-85. doi: 10.1016/j.chembiol.2006.01.004.
The ketoreductase (KR) domains eryKR(1) and eryKR(2) from the erythromycin-producing polyketide synthase (PKS) reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Modeling of eryKR(1) and eryKR(2) showed that conserved amino acids previously correlated with production of alternative alcohol configurations lie in the active site. eryKR(1) domains mutated at these positions showed an altered stereochemical outcome in reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester. The wild-type eryKR(1) domain exclusively gave the (2S, 3R)-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester, while the double mutant (F141W, P144G) gave only the (2S, 3S) isomer, a switch of the alcohol stereochemistry. Mutation of the eryKR(2) domain, in contrast, greatly increased the proportion of the wild-type (2R, 3S)-alcohol product. These data confirm the role of key residues in stereocontrol and suggest an additional way to make rational alterations in polyketide antibiotic structure.
来自红霉素产生聚酮合酶（PKS）的酮还原酶（KR）结构域eryKR(1)和eryKR(2)以相反的立体特异性还原3-酮酰基硫酯中间体。eryKR(1)和eryKR(2)的建模表明，先前与替代醇构型产生相关的保守氨基酸位于活性位点。在这些位置发生突变的eryKR(1)结构域在还原(2R,S)-2-甲基-3-氧代戊酸N-乙酰半胱氨酸硫酯时显示出立体化学结果的改变。野生型eryKR(1)结构域仅产生(2S,3R)-3-羟基-2-甲基戊酸N-乙酰半胱氨酸硫酯，而双突变体（F141W，P144G）仅产生(2S,3S)异构体，即醇立体化学的转变。相比之下，eryKR(2)结构域的突变大大增加了野生型(2R,3S)-醇产物的比例。这些数据证实了关键残基在立体控制中的作用，并提出了一种在聚酮类抗生素结构中进行合理改变的额外方法。
