Path dependence theory-inspired rational design of toad tryptophan hydroxylase 1 via engineering of non-active substrate binding pockets.

Establishing efficient 5-hydroxytryptophan (5-HTP) biosynthesis systems remains industrially challenging. Conceptually guided by Path Dependence Theory, which suggests that initial states can influence subsequent developments, we hypothesized that optimizing substrate routing away from non-active substrate binding pockets would enhance catalytic efficiency by reducing kinetic trapping. Using site-directed mutagenesis on Bufo bufo gargarizans tryptophan hydroxylase 1 (BbgTPH1), we engineered mutants Y59R-S120R and A198R to weaken substrate L-tryptophan binding to these non-active pocket. The resulting variants achieved up to an 18-fold increase in catalytic efficiency (kcat/Km), surpassing the previous maximum improvement of 4.25-fold reported for tryptophan hydroxylase. This approach provides optimized biocatalysts for 5-HTP production and establishes a conceptually informed strategy for enhancing enzyme activity through the rational engineering of non-active pockets.