Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues.
Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues.
复制标题
DOI：
10.1021/acs.biochem.6b00024
复制
发表时间：
2016-03-01
期刊：
影响因子：
2.9
通讯作者：
Cane DE
中科院分区：
文献类型：
--
作者：
Xie X;Garg A;Keatinge-Clay AT;Khosla C;Cane DE
研究了保守的活性部位酪氨酸和丝氨酸残基在聚酮合成酶酮还原酶(PKS-KR)结构域催化的异构化反应中的作用。突变和野生型的表异构体活性KR结构域，包括固有的氧化还原失活的EryKR30和PicKR30以及氧化还原失活的EryKR30突变体，与[2-2H]-(2R，3S)-2-methyl-3-hydroxypentanoyl-SACP([2-2H]-2)和0.05当量的NADP+在氧化还原活性的、差向异构体的EryKR6结构域存在的情况下孵育。用串联平衡同位素交换法测定每个突变体的残存下同分异构酶活性，用LC-MS-MS定量诊断泛影葡胺排出片段(4)的氚含量，监测2的一级、依赖于时间的同位素洗脱。单独或一起替换活性位点Tyr或Ser残基显著降低了观察到的每个KR结构域的异构体酶活性，而对底物结合的影响最小。我们的结果表明，PKS KR结构域的异构体酶和还原酶活性共享一个共同的活性部位，两个反应都利用了同一对Tyr和Ser残基。
The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR30 and PicKR30 as well as redox-inactive mutants of EryKR1, were incubated with [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-2) and 0.05 equiv of NADP+ in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by LC-MS-MS with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.