PDF(Pubmed)
Abstract：
Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.
摘要：
酶支架是通过控制多酶级联的空间组织和化学计量来提高其催化效率的新兴方法。这项研究介绍了一个新颖的工程SCAffoldingBricks家族，名为SCAB，利用共有四三肽重复序列（CTPR）结构域用于有组织的多酶系统。开发了两个SCAB系统，一种采用可逆共价二硫键的头对尾相互作用，另一个依靠非共价金属驱动的组装通过工程金属协调接口。酶直接与SCAB模块融合，在非还原环境中或通过金属存在触发组装。甲酸脱氢酶（FDH）和L-丙氨酸脱氢酶（AlaDH）的概念验证显示，与游离酶相比，比生产率提高了3.6倍。共价吻合优于金属驱动组件。这种增强可能源于高阶超分子组装和改进的NADH辅因子再生，导致更有效的级联。这项研究强调了蛋白质工程定制支架的潜力，利用超分子空间组织工具，更有效的酶级联反应。
