Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide

• DOI: 10.1016/j.ijbiomac.2023.127800
• Publication Date: 2023-11-01T01:42:25Z
• Authors: Dong Ma; Zhongyi Cheng; Laichuang Han; Junling Guo; Lukasz Peplowski; Zhemin Zhou
• Keywords: Nitrile hydratase

Cinnamamide and its derivatives are the most common important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been used large-scale industrial production of nicotinamide acrylamide, while catalytic activity is extremely low or inactive for bulky substrates such as cinnamonitrile. Therefore, beneficial variant βF37P/L48P/F51N were obtained from PtNHase Pseudonocardia thermophila JCM3095 by reshaping substrate access tunnel binding pocket, which exhibited 14.88-fold improved efficiency compared to wild-type PtNHase. Structure analysis, molecular dynamics simulations dynamical cross-correlation matrix (DCCM) analysis revealed that introduced mutations enlarged enhanced overall anti-correlated movements enzymes, would promote product release during dynamic process catalysis. In a hydration process, complete conversion 5 mM cinnamonitrile was achieved 50 mL reaction, with cinnamamide yield almost 100 % productivity 0.736 g L−1 h−1. The study demonstrates co-evolution pocket an effective strategy, provides valuable reference future research. Furthermore, NHases have huge potential catalyzing nitriles form corresponding amides production.