Engineering of human tryptophan hydroxylase 2 for efficient synthesis of 5-hydroxytryptophan

• Published in: International journal of biological macromolecules 2024-03, Vol.260 (Pt 1), p.129484-129484, Article 129484
• Main Authors: Wang, BingBing, Xu, Jian-Zhong, Liu, Shuai, Rao, Zhi-Ming, Zhang, Wei-Guo
• Format: Article
• Language: English
• Subjects: 5-Hydroxytryptophan; 5-Hydroxytryptophan - metabolism; Catalytic Domain; Humans; Hydroxylation; Molecular dynamics simulation; Semi-rational combinatorial active site saturation testing; Tryptophan - chemistry; Tryptophan hydroxylase; Tryptophan Hydroxylase - chemistry; Tryptophan Hydroxylase - genetics; Tryptophan Hydroxylase - metabolism
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.129484

L-Tryptophan hydroxylation catalyzed by tryptophan hydroxylase (TPH) presents a promising method for synthesizing 5-hydroxytryptophan (5-HTP), yet the limited activity of wild-type human TPH2 restricts its application. A high-activity mutant, MT10 (H318E/H323E), was developed through semi-rational active site saturation testing (CAST) of wild-type TPH2, exhibiting a 2.85-fold increase in kcat/Km over the wild type, thus enhancing catalytic efficiency. Two biotransformation systems were developed, including an in vitro one-pot system and a Whole-Cell Catalysis System (WCCS). In the WCCS, MT10 achieved a conversion rate of only 31.5 % within 32 h. In the one-pot reaction, MT10 converted 50 mM L-tryptophan to 44.5 mM 5-HTP within 8 h, achieving an 89 % conversion rate, outperforming the M1 (NΔ143/CΔ26) variant. Molecular dynamics simulations indicated enhanced interactions of MT10 with the substrate, suggesting improved binding affinity and system stability. This study offers an effective approach for the efficient production of 5-HTP. •Semi-rational CAST modified wild-type Hs-TPH2, creating mutant MT10 with 2.85 times higher kcat/Km.•The double mutant MT10 showed the highest 5-HTP conversion rate of 89% within 8 h.•Semi-rational mutagenesis and high-throughput screening effectively yield active Hs-TPH2 mutants with commercial potential.