Department of Crystallography & Structural Biology, Institute of Physical Chemistry "Rocasolano" , CSIC , 28006 Madrid , Spain.
Department of Biocatalysis, Institute of Catalysis , CSIC , 28049 Madrid , Spain.
ACS Chem Biol. 2018 Dec 21;13(12):3259-3268. doi: 10.1021/acschembio.8b00500. Epub 2018 Nov 16.
Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C-H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.
由于其最低要求、基质的混杂性和产物的选择性，真菌过氧化物酶现在被认为是 C-H 氧化官能化生物催化剂中的瑰宝。在这项工作中，酵母表达的第一种实验室进化过氧化物酶的晶体结构在 1.5Å 的分辨率下被确定。在从田头菇属中进化而来的过氧化物酶和天然过氧化物酶之间检测到显著的差异，包括由于定向进化而积累的突变导致完整的 N 端和更宽的血红素进入通道的存在。进一步的诱变和过氧化物酶和过氧化物酶底物的浸泡实验表明，血红素通道的动态运输是过氧化物酶异常基质混杂性的主要驱动力。因此，这项研究在原子水平上提供了第一个关于这种多功能生物催化剂的底物结合模式的结构证据，并在生物和化学背景下进行了讨论。
