Engineering the substrate channeling interface between the heterologous modules for a highly efficient FAD synthetase

Abstract

Flavin adenine dinucleotide synthetase (FADS) orchestrates the biosynthesis of flavin adenine dinucleotide (FAD) with two consecutive catalytic modules. The process entails the release and transfer of the reactant from the initial module to the subsequent one, while the transport process of the intermediate flavin mononucleotide (FMN) between these modules remains unclear. This study focuses on the oligomeric assembly of CaFADS from Corynebacterium ammoniagenes, revealing insights into substrate channeling interface between the two heterologous modules. Employing a semi-rational mutagenesis for residues in the interfaces of the heterologous modules, L204V is screened out and exhibited a 2.3-fold improvement in FAD productivity compared to the wild type. Molecular dynamics simulations indicate the L204V mutant facilitates a larger exit for the intermediate FMN, expediting its release from the first catalytic module. While the mutant showed minor changes of binding affinity with the riboflavin calculated by surface plasmon resonance assay, steered MD simulations of FMN from the first catalytic module demonstrate that the L204V mutant reduces the binding free energy for FMN departure, substantiating its role in improving substrate transport efficiency. The findings shed light on the intricate interplay between different modules of bifunctional enzymes and emphasize the importance of substrate channeling in enhancing catalytic efficiency.