Cooperative engineering of distal and proximal domains in L-alanine dehydrogenase breaks activity-stability trade-off for efficient hydroxypyruvic acid synthesis.

L-alanine dehydrogenase (L-AlaDH) demonstrates significant potential due to its high activity toward L-alanine and broad substrate spectrum. When using l-serine (L-Ser) as substrate, it generates hydroxypyruvic acid (HPA), a crucial precursor for chiral drug synthesis. However, wild-type enzymes display poor activity toward L-Ser. Unlike conventional iterative saturation mutagenesis (ISM) that typically focuses solely on proximal active sites and often suffers from the activity-stability trade-off, herein, we propose a distal-proximal co-evolution strategy. This approach first stabilizes distal cooperative domains to create a rigid scaffold, enabling subsequent proximal active-site remodeling of Mycobacterium tuberculosis L-AlaDH (mtAlaDH) for highly efficient HPA synthesis. First, structural analysis revealed critical residues in the proximal substrate-binding pocket and substrate-binding sites of mtAlaDH, while molecular dynamics simulations identified key residues in the distal cooperative region. Sequential ISM of mtAlaDH, progressing from distal-to-proximal regions, yielded variant M4 (P280M/N11R/F94Q/K75L), which displays an 11.2-fold increase in catalytic efficiency toward L-Ser and improved thermal stability (ΔTm = 4.3 °C). The enzyme maintains over 75% activity in 0.5 M ammonium, and its substrate spectrum is also broadened. Molecular dynamics simulations revealed that enhanced catalytic efficiency originated from geometrically adaptive configurations of the substrate-binding pocket and strengthened substrate binding, while structural rigidification contributed to thermal stability. Finally, a 100 mL-scale reaction using 50 mM L-Ser coupled M4 with NADH oxidase (NOX) achieved 80.2% conversion and 2.52 g/L/h productivity for HPA synthesis. This study provides an effective enzyme engineering strategy to promote biocatalytic applications in organic synthesis.