Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange.
Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange.
复制标题
DOI：
10.1021/ja5056998
复制
发表时间：
2014-07-23
影响因子：
15
通讯作者：
Cane, David E.
中科院分区：
文献类型：
--
作者：
Garg, Ashish;Xie, Xinqiang;Keatinge-Clay, Adrian;Khosla, Chaitan;Cane, David E.
许多模块化聚酮合成酶含有一个或多个功能未知的氧化还原非活性结构域，这些结构域与酮还原酶（KR）结构域高度同源。一项新开发的连续平衡同位素交换（EIX）分析已经确定，这种“KR0”结构域催化瞬态(2R)-2-甲基-3-酮酰基- acp中间体向相应的(2S)-2-甲基-3-酮酰基- acp非对映体的生物合成必需的外映体化。[2-2H]-(2R,3S)-2-甲基-3-羟基戊烷基- sacp （[2-2H]-3b）与6-脱氧红素内酯B合成酶模块3的EryKR30结构域，以及氧化还原活性的非映外化EryKR6结构域和NADP+一起培养，由于EryKR30催化了瞬时生成的[2-2H]-2-甲基-3-酮戊烷基- acp的映外化，导致3b中氘的时间和辅助因子依赖性洗涤(4)。picromycin合成酶模块3的氧化还原活性PicKR30也得到了类似的结果。通过诱变epimase -active EryKR1酶的NADPH结合位点，构建了四个氧化还原无活性突变体。Tandem EIX证实，这些EryKR10突变体保留了亲本EryKR1结构域固有的表观酶活性。这些结果确定了氧化还原无活性的KR0结构域的内在外链酶活性，排除了NADPH辅助因子在外链异构化中的任何作用，并为解耦大量PKS结构域的外链酶和还原酶活性提供了一般的实验基础。
Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such “KR0” domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-3b) with the EryKR30 domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP+ resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR30-catalyzed epimerization of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR30 from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR10 mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR0 domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
登录
查看更多内容
影响因子：
4
作者：
Zheng, Jianting;Piasecki, Shawn K.;Keatinge-Clay, Adrian T.
通讯作者：
Keatinge-Clay, Adrian T.
影响因子：
5.7
作者：
Zheng, Jianting;Taylor, Clint A.;Keatinge-Clay, Adrian T.
通讯作者：
Keatinge-Clay, Adrian T.
影响因子：
5.7
作者：
Keatinge-Clay, AT;Stroud, RM
通讯作者：
Stroud, RM
影响因子：
64.8
作者：
CORTES, J;HAYDOCK, SF;LEADLAY, PF
通讯作者：
LEADLAY, PF
影响因子：
2.7
作者：
Meluzzi, Dario;Zheng, Wei Hao;Dorrestein, Pieter C.
通讯作者：
Dorrestein, Pieter C.