Torsion-spring hypothesis guided pocket-hinge synergistic engineering of glutamate dehydrogenase for efficient synthesis of unnatural amino acids.

Glutamate dehydrogenase (GluDH) is a promising biocatalyst for the asymmetric synthesis of unnatural amino acids, but NADH-dependent GluDH is generally limited by low activity toward non-natural substrates. Herein, we engineered the GluDH from Clostridium difficile 630 (CdGluDH) to overcome these limitations. Molecular dynamics (MD) simulations revealed that CdGluDH undergoes torsion-spring-like conformational transitions, characterized by helical hinge deformation and twisting of two lever arms. The twisting frequency of the lever arms is influenced by both external forces and intrinsic stress of helical coil. We therefore propose a torsional spring hypothesis: the concomitant enhancement of ligand affinity in the substrate-binding pocket and modulation of hinge rigidity reduces unproductive conformations and lowers the energy barrier, thus accelerating conformational transition and boosting the catalytic rate. Guided by this hypothesis, we performed pocket-hinge synergy engineering to design CdGluDH. Site-directed saturation mutagenesis identified two pivotal pocket residues, V143 and A145. The resulting A145G/V143G mutant increased specific activity toward the model substrate 2-oxo-4-[(hydroxy)(methyl)phosphinyl]butyric acid from 0.14 U/mg to 137.42 U/mg. Subsequent iterative saturation mutagenesis in the hinge region yielded mutant A145G/V143G/K22I/Y395A/E401T/T398S (GPG-B4M), which exhibited a specific activity of 278.94 U/mg, representing a 1992.4-fold enhancement over the wild-type enzyme. MD simulations validated the mechanism of the torsion-spring hypothesis. The engineered mutants exhibited broad substrate promiscuity and enabled efficient synthesis of diverse unnatural amino acids. This work provides a potentially general pocket-hinge synergistic engineering framework for the rational design of allosteric enzymes.