Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domains via diffusible, synthetic substrates.
Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domains via diffusible, synthetic substrates.
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DOI：
10.1039/c5sc01505g
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发表时间：
2015-08-14
期刊：
影响因子：
8.4
通讯作者：
Aldrich CC
中科院分区：
文献类型：
--
作者：
Fiers WD;Dodge GJ;Li Y;Smith JL;Fecik RA;Aldrich CC
天然和改性的基板加上LC-MS/MS分析的产品揭示了立体特异性和立体选择性的聚酮化合物双结构域。聚酮化合物合成酶（PKS）β-加工结构域负责聚酮化合物天然产物的许多立体化学复杂性。虽然β-加工结构域的重要性已被充分注意和显著探索，但与隐蔽立体化学和远程立体中心的影响有关的关键立体化学细节尚未完全辨别。为了揭示来自泰乐菌素途径的酮还原酶（KR）和脱氢酶（DH）的内部工作，重组表达由TylDH 3-KR 3组成的双结构域，并用全长四酮化合物底物询问以探测邻位和远端立体化学的影响。体外产物分离分析揭示了隐藏的KR作为d-醇的产物和DH作为反式烯烃的产物。脱水反应的稳态动力学分析表明，在β-位上具有严格的立体化学耐受性，因为d-构型底物的处理效率比l-醇高100倍以上。出乎意料的是，当远侧的ε-和ε-立构中心反转时，k cat/K M值减小14- 45倍。预测这种立体化学区分是由可能不利于ε-差向异构体结合的烯丙基A1，3菌株的组合以及与ε-差向异构体的静电相互作用的损失驱动的。我们的研究结果强烈表明，PKS酶可能发挥作用，通过其底物立体特异性，磨练最终PKS产品的构型纯度，在完善的立体化学结果的前面的模块。
Natural and modified substrates coupled with LC-MS/MS analysis of products revealed the stereospecificity and stereoselectivity of a polyketide didomain. Polyketide synthase (PKS) β-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of β-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry. In vitro product isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH as trans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the β-position as d-configured substrates were processed more than 100 times more efficiently than l-alcohols. Unexpectedly, the k cat/K M values were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination is predicted to be driven by a combination of allylic A1,3 strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.