Dehydrogenase versus Oxidase Function: The Interplay between Substrate Binding and Flavin Microenvironment

Redox enzymes, mostly equipped with metal or organic cofactors, can vary their reactivity with oxygen by orders of magnitude. Understanding how oxygen reactivity is controlled by the protein milieu remains an open issue, with broad implications for mechanistic enzymology and enzyme design. Here, we address this problem by focusing on a widespread group of flavoenzymes that oxidize phenolic compounds derived from microbial lignin degradation, using either oxygen or cytochrome c as an electron acceptor. A comprehensive phylogenetic analysis revealed conserved amino acid motifs in the flavin-binding site. Using a combination of kinetic, mutagenesis, structural, and computational methods, we examined the role of these residues. Our results demonstrate that subtle and localized changes in the flavin environment can drastically impact oxygen reactivity. These effects are afforded through the creation or blockade of pathways for oxygen diffusion. Substrate binding plays a crucial role by potentially obstructing oxygen access to the flavin, thus influencing the enzyme’s reactivity. The switch between oxidase and dehydrogenase functionalities is thereby achieved through targeted, site-specific amino acid replacements that finely tune the microenvironment around the flavin. Our findings explain how very similar enzymes can exhibit distinct functional properties, operating as oxidases or dehydrogenases. They further provide valuable insights for the rational design and engineering of enzymes with tailored functions.多数氧化还原酶（redox enzyme）携带有金属或有机辅因子，其与氧气的反应活性可在数个数量级范围内变动。阐明蛋白质微环境如何调控酶的氧反应活性，仍是一个未解难题，该问题对酶学机制研究与酶理性设计均具有重要意义。本研究聚焦于一类广泛存在的黄素酶（flavoenzyme），这类酶可利用氧气或细胞色素c（cytochrome c）作为电子受体，氧化源自微生物木质素降解过程的酚类化合物，以此解决上述科学问题。通过全面的系统发育分析，我们在黄素结合位点（flavin-binding site）中发现了保守氨基酸基序。我们结合动力学实验、诱变技术、结构生物学分析与计算方法，探究了这些氨基酸残基的功能作用。研究结果表明，黄素微环境中细微且局部的变化，可对酶的氧反应活性产生显著影响。这类影响通过构建或阻断氧气扩散通路得以实现。底物结合可通过阻断氧气抵达黄素结合位点的路径，对酶的反应活性产生关键调控作用。因此，通过靶向性的位点特异性氨基酸替换，精细调控黄素周围的微环境，即可实现氧化酶与脱氢酶功能之间的切换。本研究阐明了结构高度相似的酶为何可展现出截然不同的功能特性——可作为氧化酶或脱氢酶发挥催化作用。此外，本研究还为定制化功能酶的理性设计与工程改造提供了重要的理论参考。