The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure-function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)-Tyr170 (general base)-Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2',2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure-function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

中文翻译：

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洛伐他汀水解酶催化机理的结构见解。

产自青霉青霉的洛伐他汀水解酶PcEST在一步合成莫纳可林J的工业规模应用中显示出巨大的工业潜力，莫纳可林J是合成降胆固醇药物辛伐他汀的关键前体。该酶特异性和有效地催化洛伐他汀向莫纳可林J的转化，但不能水解辛伐他汀。因此，了解PcEST的催化机理和结构-功能关系对于进一步的洛伐他汀水解酶的筛选，工程和商业应用非常重要。在这里，我们解决了四个X射线晶体结构，包括apo PcEST（2.3Å），与莫纳可林J复合的PcEST（2.48Å），与底物类似物辛伐他汀（2.4Å）复合的PcEST，以及灭活的PcEST变体（S57A）洛伐他汀底物（2.3Å）。基于结构的生化分析和诱变分析表明，Ser57（亲核试剂）-Tyr170（通用碱基）-Lys60（通用酸）催化三联体，活性位点周围的氢键网络（Trp344和Tyr127）以及特定的底物结合通道共同决定了PcEST的有效和特异性洛伐他汀水解作用。此外，由辛伐他汀的2'，2-二甲基丁酰基引起的对亲核攻击的空间效应导致PcEST对辛伐他汀没有活性。在结构比较的基础上，我们提出了几种指标来定义洛伐他汀酯酶。此外，使用结构指导的酶工程技术，我们开发了具有改善的溶解度和热稳定性的PcEST变体D106A，表明该变体在工业过程中的应用前景广阔。据我们所知，