Structure-Guided Engineering of Tryptophan Hydroxylase Relieves Tunnel Congestion and Substrate Trapping to Enhance 5-Hydroxytryptophan Biosynthesis

Substrate inhibition limits the industrial use of tryptophan hydroxylase (TPH), the key catalyst for 5-hydroxytryptophan (5-HTP) production, by causing tunnel congestion and substrate trapping at high concentrations. We developed a mechanism-guided strategy to overcome this. The crystal structure of the Y235S (MS) variant revealed a 243% expansion of the substrate channel, reducing tunnel congestion and increasing activity 2.38-fold, though substrate affinity decreased. Mechanistic analysis showed loop II acts as a molecular gate controlling cofactor-substrate binding. Its rational stabilization in variant MS4 enhanced loop stability and optimized substrate orientation, increasing catalytic efficiency by over 150% compared to MS and specific activity by 285% compared to wild-type. This approach proved generalizable across TPH orthologs. Combined with a tetrahydrobiopterin regeneration system, MS4 broke through the substrate concentration limitation, achieving >5-fold higher whole-cell 5-HTP production (16.37 mM in 4 h). This work establishes a general framework for relieving tunnel congestion and substrate trapping through integrated structural, computational, and loop engineering.