Institute of Physical Chemistry, Department of Chemistry, University of Basel, 4058, Basel, Switzerland.
Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland.
Nat Commun. 2024 Feb 28;15(1):1807. doi: 10.1038/s41467-024-45630-3.
Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We use enzyme proximity sequencing to analyze how 6399 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase from Rhodotorula gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. Enzyme proximity sequencing can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.
理解酶序列、折叠稳定性和催化活性之间的复杂关系对于工业和生物医学中的应用至关重要。然而，目前的酶分析技术受到限制，无法同时解析稳定性和活性表型，并将其与大规模基因序列联系起来。在这里，我们提出了酶邻近测序的发展，这是一种深度突变扫描方法，利用过氧化物酶介导的自由基标记，以单细胞保真度来剖析数千个突变对氧化还原酶在单个实验中的稳定性和催化活性的影响。我们使用酶邻近测序来分析 6399 个错义突变如何影响来自 Rhodotorula gracilis 的 D-氨基酸氧化酶的折叠稳定性和催化活性。所得数据集表明，在自然进化过程中，活性限制了折叠稳定性，并确定了远离活性位点的热点作为候选突变，这些突变可以提高催化活性而不牺牲稳定性。酶邻近测序可以扩展到其他酶类，并为酶结构和功能的生物物理原理提供有价值的见解。
