Structure-Guided Remodeling of Multi-Substrate Binding Pocket in Nicotinamide Mononucleotide Adenylyltransferase for Efficient NAD+ Synthesis

The rational engineering of enzymes that coordinate multiple substrates within a confined active site remains a major challenge in biocatalysis. Here, we present a structure-guided dual-cluster engineering strategy that exploits low epistatic coupling among conserved residues to independently tune electronic and geometric active-site features while avoiding trade-offs. This approach was validated through re-engineering Methanothermobacter thermautotrophicus nicotinamide mononucleotide adenylyltransferase. The optimal triple mutant MT3 (V6C/Q10E/P11T) showed a 5.1-fold increase in catalytic efficiency (kcat/Km) over wild type. Molecular dynamics simulations revealed that the mutations synergistically compact the substrate-binding pocket, reduce the catalytic distance by 1.4 Å, and reshape local electrostatics to stabilize a near-attack conformation, thereby lowering the reaction barrier. In a 30-L bioreactor, MT3 achieved 85.3% conversion and a titer of 112.5 g L–1 NAD+ within 8 h, underscoring its industrial potential. Collectively, this study provides a transferable framework for engineering complex multisubstrate enzymes by integrating structural dynamics with process-scale validation.