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首页> 外文期刊>Applied Microbiology >Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis
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Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

机译：通过突变分析定位嗜热双歧杆菌苯丙酮单加氧酶的底物结合位点

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摘要

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.
机译：Baeyer-Villiger单加氧酶催化生物催化应用中感兴趣的氧化反应。在这些酶中，来自Thermobifida fusca的苯丙酮单加氧酶（PAMO）是唯一显示出显着稳定性的蛋白质。尽管相关的酶通常具有广泛的底物范围，但PAMO仅接受有限数量的底物。由于在PAMO阐明的晶体结构中不存在底物，因此尚未定义该蛋白质的底物结合位点。在这项研究中，已制备出作用于多种化合物的环戊酮单加氧酶的结构模型，并与PAMO的结构进行了比较。这揭示了PAMO活性位点中的15个氨基酸位置，这可能解释了其相对窄的底物特异性。我们设计和分析了30个单突变体和多突变体，以验证这些位置的作用。广泛的底物筛选显示了几个突变体，这些突变体在Baeyer-Villiger反应和硫氧化反应中显示出增强的活性并改变了区域或对映体选择性。进一步的底物谱分析导致鉴定出对合成上有吸引力的化合物具有改进的催化性能的突变体。而且，与野生型酶相比，突变体的热稳定性没有受到损害。我们的数据表明，在PAMO活性位点（即V54，I67，Q152和A435）中确定的位置有助于该酶的底物特异性。这些发现将有助于PAMO和相关单加氧酶的更加专门和有效的重新设计，以扩大底物范围。

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