Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Appl Environ Microbiol. 2011 Aug 15;77(16):5730-8. doi: 10.1128/AEM.00687-11. Epub 2011 Jul 1.
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.
过氧化物单加氧酶催化的氧化反应在生物催化应用中具有重要意义。在这些酶中，热纤梭菌来源的苯乙酮单加氧酶（PAMO）是唯一一种表现出显著稳定性的蛋白质。虽然相关酶通常具有广泛的底物谱，但 PAMO 仅接受有限数量的底物。由于在 PAMO 的阐明晶体结构中不存在底物，因此该蛋白质的底物结合位点尚未确定。在这项研究中，制备了作用于广泛化合物的环戊酮单加氧酶的结构模型，并将其与 PAMO 的结构进行了比较。这揭示了 PAMO 活性位点中 15 个氨基酸位置，这些位置可能与其相对较窄的底物特异性有关。我们设计并分析了 30 个单突变体和多个突变体，以验证这些位置的作用。广泛的底物筛选揭示了一些突变体，它们在 Baeyer-Villiger 反应和磺氧化反应中表现出增加的活性和改变的区域或对映选择性。进一步的底物分析导致鉴定出对具有吸引力的合成化合物具有改进催化性能的突变体。此外，与野生型酶相比，突变体的热稳定性没有受到影响。我们的数据表明，在 PAMO 的活性位点内鉴定出的位置，即 V54、I67、Q152 和 A435，对该酶的底物特异性有贡献。这些发现将有助于更有针对性和有效地重新设计 PAMO 和相关单加氧酶，以扩大其底物范围。
