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Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
Frontiers in Bioengineering and Biotechnology ( IF 4.3 ) Pub Date : 2021-09-14 , DOI: 10.3389/fbioe.2021.741282
Patricia Molina-Espeja 1 , Alejandro Beltran-Nogal 1 , Maria Alejandra Alfuzzi 1 , Victor Guallar 2, 3 , Miguel Alcalde 1
Affiliation  

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.



中文翻译:

绘制来自 Agrocybe aegerita 的进化真菌过氧化酶中过氧化活性的潜在决定因素

真菌非特异性过氧化酶 (UPO) 是具有过氧化活性的混合生物催化剂,可将氧气插入未活化的化合物中,同时还具有单电子氧化反应的收敛过氧化活性。在几种木质素分解过氧化物酶中,过氧化活性位点与蛋白质表面的可氧化芳香残基相关,该残基通过长程电子转移 (LRET) 途径连接到隐藏的血红素结构域。然而,这些酶的过氧化活性也可能在血红素通路上启动。在这项研究中,我们使用进化的分泌变体(PaDa-I 突变体)检查了 UPO 过氧化活性的起源杨树菇作为我们的出发点。在通过 QM/MM 分析 PaDa-I 表面潜在的形成自由基的芳香残基后,构建了 Trp24、Tyr47、Tyr79、Tyr151、Tyr265、Tyr281、Tyr293 和 Tyr325 的独立饱和诱变文库,并使用过氧化和过氧化底物进行筛选。这些突变体文库大多无活性,仅检测到少数功能性克隆,没有一个在过氧化和过氧化活性方面显示出显着差异。相比之下,当在血红素通道外入口处发现的灵活 Gly314-Gly318 环进行组合饱和诱变和计算分析时,发现了具有改进动力学和过氧化底物 pH 活性曲线变化的突变体,而它们保留了过氧化底物的动力学值。尽管变体携带多达四个连续突变,但这种惊人的变化伴随着动力学热稳定性提高了 4.5°C。总之,我们的研究证明,与迄今为止描述的其他木质素分解过氧化物酶不同,UPO 中过氧化活性的起源不依赖于来自蛋白质表面可氧化残基的 LRET 途径,而是它似乎仅位于血红素访问通道。

更新日期:2021-09-14
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