9WGZ

Tryptophan hydroxylase mutant - Y235S

Summary for 9WGZ

• Entry DOI: 10.2210/pdb9wgz/pdb
• Descriptor: Tryptophan 5-hydroxylase 1, FE (III) ION (3 entities in total)
• Functional Keywords: mutant, oxidoreductase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 37048.06

Authors

Han, J.,Liu, F.,Rao, Z.M. (deposition date: 2025-08-25, release date: 2026-02-11)

Primary citation

Han, J.,You, J.,Fan, Z.,Liu, F.,Zhang, H.,Zhu, R.,Chen, X.,Zhang, R.,Rao, Z.
Structure-Guided Engineering of Tryptophan Hydroxylase Relieves Tunnel Congestion and Substrate Trapping to Enhance 5-Hydroxytryptophan Biosynthesis.
J.Agric.Food Chem., 74:1312-1325, 2026

PubMed Abstract: 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.

PubMed: 41460668
DOI: 10.1021/acs.jafc.5c14467

Experimental method

X-RAY DIFFRACTION (1.857 Å)