Garg Ashish, Xie Xinqiang, Keatinge-Clay Adrian, Khosla Chaitan, Cane David E
Department of Chemistry, Box H, Brown University , Providence, Rhode Island 02912-9108, United States.
J Am Chem Soc. 2014 Jul 23;136(29):10190-3. doi: 10.1021/ja5056998. Epub 2014 Jul 10.
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 "KR(0)" 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-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) 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 EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) 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 EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) 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.
许多模块化聚酮合酶含有一个或多个功能未知的氧化还原无活性结构域，这些结构域与酮还原酶（KR）结构域高度同源。一种新开发的串联平衡同位素交换（EIX）分析方法现已证实，此类“KR(0)”结构域催化瞬时（2R）-2-甲基-3-酮酰基-ACP中间体生物合成必需的差向异构化反应，生成相应的（2S）-2-甲基-3-酮酰基-ACP非对映异构体。将[2-（2）H]-（2R，3S）-2-甲基-3-羟基戊酰-SACP（[2-（2）H]-3b）与6-脱氧红霉内酯B合酶模块3中的EryKR3(0)结构域、具有氧化还原活性且无差向异构化作用的EryKR6结构域以及NADP(+)一起孵育，由于EryKR3(0)催化瞬时生成的[2-（2）H]-2-甲基-3-酮戊酰-ACP（4）发生差向异构化反应，则导致3b中的氘随时间和辅因子依赖性地洗脱。对巨霉素合酶模块3中的氧化还原无活性PicKR3(0)也得到了类似结果。通过对该酶的NADPH结合位点进行诱变，构建了4个具有差向异构酶活性的EryKR1的氧化还原无活性突变体。串联EIX分析证实，这些EryKR1(0)突变体保留了亲本EryKR1结构域的固有差向异构酶活性。这些结果证实了氧化还原无活性KR(0)结构域的固有差向异构酶活性，排除了NADPH辅因子在差向异构化反应中的任何作用，并为区分一大类聚酮合酶结构域的差向异构酶和还原酶活性提供了通用的实验基础。
