Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase
Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase
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DOI：
10.1016/j.chembiol.2006.01.004
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发表时间：
2006-03-01
影响因子：
--
通讯作者：
Leadlay, PF
中科院分区：
文献类型：
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作者：
Baerga-Ortiz, A;Popovic, B;Leadlay, PF
产红霉素聚酮合成酶(PKS)的酮还原酶(KR)结构域eryKR(1)和eryKR(2)还原3-酮酰基硫酸酯中间体，具有相反的立体特异性。对eryKR(1)和eryKR(2)的模拟表明，以前与替代醇构型产生相关的保守氨基酸位于活性部位。在这些位置突变的erKR(1)结构域在(2R，S)-2-甲基-3-氧戊酸N-乙酰半胱胺硫酸酯的还原中显示出改变的立体化学结果。野生型eryKR(1)结构域只产生(2S，3R)-3-羟基-2-甲基戊酸N-乙酰半胱胺硫酯，而双突变体(F141W，P144G)只给出(2S，3S)异构体，这是酒精立体化学的开关。相反，eryKR(2)结构域的突变大大增加了野生型(2R，3S)-酒精产物的比例。这些数据证实了关键残基在立体控制中的作用，并提出了另一种合理改变聚酮抗生素结构的方法。
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.