Wang Yang, Douglas Trevor
Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States.
ACS Appl Mater Interfaces. 2023 Sep 20;15(37):43621-43632. doi: 10.1021/acsami.3c10340. Epub 2023 Sep 11.
Spatial organization of biocatalytic activities is crucial to organisms to efficiently process complex metabolism. Inspired by this mechanism, artificial scaffold structures are designed to harbor functionally coupled biocatalysts, resulting in acellular materials that can complete multistep reactions at high efficiency and low cost. Substrate channeling is an approach for efficiency enhancement of multistep reactions, but fast diffusion of small molecule intermediates poses a major challenge to achieve channeling in vitro. Here, we explore how multistep biocatalysis is affected, and can be modulated, by cofactor-enzyme colocalization within a synthetic bioinspired material. In this material, a heterogeneous protein macromolecular framework (PMF) acts as a porous host matrix for colocalization of two coupled enzymes and their small molecule cofactor, nicotinamide adenine dinucleotide (NAD). After formation of the PMF from a higher order assembly of P22 virus-like particles (VLPs), the enzymes were partitioned into the PMF by covalent attachment and presentation on the VLP exterior. Using a collective property of the PMF (i.e., high density of negative charges in the PMF), NAD molecules were partitioned into the framework via electrostatic interactions after being conjugated to a polycationic species. This effectively controlled the localization and diffusion of NAD, resulting in substrate channeling between the enzymes. Changing ionic strength modulates the PMF-NAD interactions, tuning two properties that impact the multistep efficiency oppositely in response to ionic strength: cofactor partitioning (colocalization with the enzymes) and cofactor mobility (translocation between the enzymes). Within the range tested, we observed a maximum of 5-fold increase or 75% decrease in multistep efficiency as compared to free enzymes in solution, which suggest both the colocalization and the mobility are critical for the multistep efficiency. This work demonstrates utility of collective behaviors, exhibited by hierarchical bioassemblies, in the construction of functional materials for enzyme cascades, which possess properties such as tunable multistep biocatalysis.
生物催化活性的空间组织对于生物体高效处理复杂代谢过程至关重要。受此机制启发，人们设计了人工支架结构来容纳功能耦合的生物催化剂，从而得到能够以高效率和低成本完成多步反应的无细胞材料。底物通道化是提高多步反应效率的一种方法，但小分子中间体的快速扩散对在体外实现通道化构成了重大挑战。在此，我们探讨了在一种合成的仿生材料中，辅因子与酶的共定位如何影响多步生物催化以及如何对其进行调控。在这种材料中，一种异质蛋白质大分子框架（PMF）作为多孔宿主基质，用于两种耦合酶及其小分子辅因子烟酰胺腺嘌呤二核苷酸（NAD）的共定位。由P22病毒样颗粒（VLP）的高阶组装形成PMF后，通过共价连接并呈现在VLP外部，将酶分配到PMF中。利用PMF的一种集体性质（即PMF中高密度的负电荷），NAD分子在与聚阳离子物种缀合后，通过静电相互作用被分配到框架中。这有效地控制了NAD的定位和扩散，从而实现了酶之间的底物通道化。改变离子强度可调节PMF与NAD的相互作用，调节两种对多步效率有相反影响的性质以响应离子强度：辅因子分配（与酶的共定位）和辅因子流动性（酶之间的转运）。在测试范围内，我们观察到与溶液中的游离酶相比，多步效率最多可提高5倍或降低75%，这表明共定位和流动性对于多步效率都至关重要。这项工作展示了分级生物组装所表现出的集体行为在构建用于酶级联反应的功能材料中的实用性，这些材料具有可调谐的多步生物催化等特性。
