Presentation Information
[P01-038]From Pigment Biosynthesis to Neurochemical Sensing: Functional Rewiring of a Plant Dioxygenase
○Chun-Hua Hsu1 (1. National Taiwan University (Taiwan))
Keywords:
DOPA dioxygenase,Structural Biochemistry,Biophysics,Plant pigment,Biosensing
[Purpose]
4,5-DOPA extradiol dioxygenase (DOD) catalyzes the key ring-cleavage step in betalain biosynthesis by converting L-DOPA into betalamic acid, the common chromophore of betalains. In this study, we aimed to understand the structural basis of plant DOD catalysis and to explore whether this enzyme scaffold can be functionally rewired for catecholamine-related sensing applications, especially for L-DOPA and dopamine.
[Method]
We combined biochemical characterization, product analysis, X-ray crystallography, SAXS, molecular modeling, and structure-guided mutagenesis to study DOD enzymes from betalain-producing plants. Beta vulgaris DOD (BvDOD) was used as the structural prototype. In parallel, DOD-based colorimetric sensing of L-DOPA was used as a functional reference platform. Building on these results, Mirabilis jalapa DOD (MjDOD) and engineered variants were evaluated by kinetic analysis and whole-cell colorimetric screening to identify mutants with altered substrate preference.
[Results]
BvDOD was confirmed to produce betalamic acid, and structural analysis showed that it adopts an extradiol dioxygenase-like fold with a nonheme iron center coordinated by His15, His53, and His229. Mutational and modeling studies further identified His119 and His175 as important catalytic residues and supported a mechanism for L-DOPA ring cleavage. Earlier DOD-based sensing work demonstrated that this reaction can be translated into a simple optical readout for L-DOPA detection. Extending this idea, our current engineering results show that substrate selectivity in plant DODs can be shifted, and MjDOD variants with improved dopamine-responsive behavior can be identified through structure-guided mutagenesis and screening.
[Consideration]
These results suggest that plant DODs are more than pigment biosynthetic enzymes. They can also serve as tunable biocatalysts whose substrate preference and output signal can be redirected by protein engineering. The combination of structure, simulation, and screening provides a useful strategy for linking pocket architecture to catalytic selectivity and sensor function.
[Conclusion]
Our work establishes a structure-guided platform that connects betalain biosynthesis with neurochemical sensing. By defining the catalytic framework of BvDOD and extending DOD engineering toward dopamine-responsive variants, we highlight the potential of plant dioxygenases as rewritable tools for natural pigment synthesis, , and synthetic biology applications.
4,5-DOPA extradiol dioxygenase (DOD) catalyzes the key ring-cleavage step in betalain biosynthesis by converting L-DOPA into betalamic acid, the common chromophore of betalains. In this study, we aimed to understand the structural basis of plant DOD catalysis and to explore whether this enzyme scaffold can be functionally rewired for catecholamine-related sensing applications, especially for L-DOPA and dopamine.
[Method]
We combined biochemical characterization, product analysis, X-ray crystallography, SAXS, molecular modeling, and structure-guided mutagenesis to study DOD enzymes from betalain-producing plants. Beta vulgaris DOD (BvDOD) was used as the structural prototype. In parallel, DOD-based colorimetric sensing of L-DOPA was used as a functional reference platform. Building on these results, Mirabilis jalapa DOD (MjDOD) and engineered variants were evaluated by kinetic analysis and whole-cell colorimetric screening to identify mutants with altered substrate preference.
[Results]
BvDOD was confirmed to produce betalamic acid, and structural analysis showed that it adopts an extradiol dioxygenase-like fold with a nonheme iron center coordinated by His15, His53, and His229. Mutational and modeling studies further identified His119 and His175 as important catalytic residues and supported a mechanism for L-DOPA ring cleavage. Earlier DOD-based sensing work demonstrated that this reaction can be translated into a simple optical readout for L-DOPA detection. Extending this idea, our current engineering results show that substrate selectivity in plant DODs can be shifted, and MjDOD variants with improved dopamine-responsive behavior can be identified through structure-guided mutagenesis and screening.
[Consideration]
These results suggest that plant DODs are more than pigment biosynthetic enzymes. They can also serve as tunable biocatalysts whose substrate preference and output signal can be redirected by protein engineering. The combination of structure, simulation, and screening provides a useful strategy for linking pocket architecture to catalytic selectivity and sensor function.
[Conclusion]
Our work establishes a structure-guided platform that connects betalain biosynthesis with neurochemical sensing. By defining the catalytic framework of BvDOD and extending DOD engineering toward dopamine-responsive variants, we highlight the potential of plant dioxygenases as rewritable tools for natural pigment synthesis, , and synthetic biology applications.
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