Time-Resolved Studies of the Mononuclear Non-Heme Iron Enzyme Tryptophan Hydroxylase
Advisor(s)
Dr. Katherine Davis, Emory University
Confirmation
1
Document Type
Poster
Location
ONU McIntosh Center; Activities Room
Start Date
24-4-2026 12:00 PM
End Date
24-4-2026 12:50 PM
Abstract
Tryptophan hydroxylase (TPH) is a non-heme iron enzyme that catalyzes the rate-limiting conversion of tryptophan to 5-hydroxytryptophan in serotonin biosynthesis. It is the least studied of the aromatic amino acid hydroxylases and exists in two isoforms: TPH1, which functions primarily in the gut, and TPH2, which operates in the brain. Spectroscopic studies of TPH2 suggest that its cofactor, tetrahydrobioptern (BH4), directly coordinates to the iron center upon complexation with the tryptophan substrate. Interaction with molecular oxygen subsequently forms a peroxo intermediate, immediately followed by a highly reactive iron(IV)-oxo species. Due to their transient nature, these intermediates have not been structurally characterized. While TPH1 crystallizes readily, its hybrid mechanism complicates spectroscopic characterization. TPH2, by contrast, displays well-defined spectroscopic signatures, but has been more recalcitrant to crystallization efforts. The goal of this summer was to purify and crystallize TPH2 in order to study the above intermediates. TPH2 was successfully purified via a combination of immobilized metal affinity, anion exchange, and size exclusion chromatography. Over 5,000 crystallization conditions were subsequently screened varying buffer composition, protein concentrations, and oxygen availability. Although no crystals were conclusively produced, these efforts greatly improved the protein’s stability, as evidenced by decreased protein crash in both stock solutions and in crystallization conditions. Further work will focus on optimizing purification conditions to enhance crystallization and initiating catalysis in crystallo.
Recommended Citation
Pacek, Emily Joy; DeLong, Gabe; and Davis, Katherine, "Time-Resolved Studies of the Mononuclear Non-Heme Iron Enzyme Tryptophan Hydroxylase" (2026). ONU Student Research Colloquium. 65.
https://digitalcommons.onu.edu/student_research_colloquium/2026/Posters/65
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Time-Resolved Studies of the Mononuclear Non-Heme Iron Enzyme Tryptophan Hydroxylase
ONU McIntosh Center; Activities Room
Tryptophan hydroxylase (TPH) is a non-heme iron enzyme that catalyzes the rate-limiting conversion of tryptophan to 5-hydroxytryptophan in serotonin biosynthesis. It is the least studied of the aromatic amino acid hydroxylases and exists in two isoforms: TPH1, which functions primarily in the gut, and TPH2, which operates in the brain. Spectroscopic studies of TPH2 suggest that its cofactor, tetrahydrobioptern (BH4), directly coordinates to the iron center upon complexation with the tryptophan substrate. Interaction with molecular oxygen subsequently forms a peroxo intermediate, immediately followed by a highly reactive iron(IV)-oxo species. Due to their transient nature, these intermediates have not been structurally characterized. While TPH1 crystallizes readily, its hybrid mechanism complicates spectroscopic characterization. TPH2, by contrast, displays well-defined spectroscopic signatures, but has been more recalcitrant to crystallization efforts. The goal of this summer was to purify and crystallize TPH2 in order to study the above intermediates. TPH2 was successfully purified via a combination of immobilized metal affinity, anion exchange, and size exclusion chromatography. Over 5,000 crystallization conditions were subsequently screened varying buffer composition, protein concentrations, and oxygen availability. Although no crystals were conclusively produced, these efforts greatly improved the protein’s stability, as evidenced by decreased protein crash in both stock solutions and in crystallization conditions. Further work will focus on optimizing purification conditions to enhance crystallization and initiating catalysis in crystallo.