The enzyme 4,5-DOPA-extradiol-dioxygenase (DODA) represents a distinct class of extradiol dioxygenases. DODA catalyzes the cleavage of the aromatic ring in L-DOPA, leading to the formation of betalamic acid, the universal chromophore of betalain pigments.1 These pigments have garnered significant attention due to their potential as natural colorants, their beneficial effects on human health and a protective role in plants.2,3
While extradiol dioxygenases are well-characterized in microbial systems, the structure and catalytic mechanism of plant-type DODAs remain unexplored. We present a kinetic and mechanistic characterization of Beta vulgaris DODA (BvDODA), providing key insights into its catalytic operation.
To assess the stability of the purified protein, fluorescence thermal shift (FTS) analysis was performed, revealing that BvDODA exhibits the highest stability in buffers with pH 7.5–8.0. The kinetic parameters of BvDODA were determined by Michaelis–Menten model. The enzyme exhibits optimal activity at pH 6.5. The reaction follows a substrate inhibition model suggesting a mechanism that limits enzymatic activity at high substrate concentration.
Mössbauer spectroscopy data provides insights into the binding mode of the L-DOPA molecule, which plays a crucial role in the enzymatic function. Additionally, electron paramagnetic resonance (EPR) spectroscopy was performed on metal-substituted enzyme variants containing manganese and cobalt.
Five BvDODA mutants were obtained with different substrate-binding residues mutated to alanine. This site-directed mutagenesis allowed us to evaluate their impact on enzymatic activity, revealing amino acids essential for catalysis.
Our comprehensive analysis combining biochemical and spectroscopic techniques has provided insights into the substrate recognition and catalytic mechanisms of BvDODA, paving the way for the development of enzymes with enhanced betalain production capabilities.