Structural and Mechanistic Insight into Oxidative Ring Opening Catalyzed by L-DOPA Extradiol Dioxygenase from Beta vulgaris

Abstract Betalain pigments replace anthocyanins in the majority of plants from the order Caryophyllales, but they are also present in some fungi and bacteria. Their biosynthesis attracts considerable scientific interest due to their applications across various fields and also because plant pigmentation is used to study complex evolution within Caryophyllales. The key step in betalain biosynthesis is the oxidative ring opening of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by L-DOPA extradiol dioxygenase (DODA, EC: 1.13.11.29), which yields 4,5-seco-DOPA; the latter cyclizes to betalamic acid, the chromophore of all betalains. Betalain-producing plants feature two to six homologues of DODA, yet only one of them is highly active and participates in the betalain biosynthesis. Here, we report the crystal structures of the highly active DODAα1 from Beta vulgaris (beetroot), the main commercial source of betalain pigments. High-resolution structures of the apo form and complexes with L-DOPA and three substrate analogues (4-nitrocatechol, 6-nitro-L-DOPA, mimosine) provide insights into the key interactions responsible for effective substrate recognition by the enzyme. Enzymatic reaction kinetics for WT BvDODAα1 show that the enzyme is three orders of magnitude more efficient than the previously studied homologue BvDODAα2, with a pH optimum of 6.5, and that the kinetics are affected by modest substrate inhibition. The residues selected based on X-ray structures for site-directed mutagenesis, except Asp81, are critical for efficient catalysis, whereas molecular dynamics simulations show that they form strong direct and through-water contacts with the L-DOPA amino acid side chain. Mössbauer spectroscopy, supported by DFT calculations, suggests that the catechol ring of L-DOPA binds to the enzyme with both hydroxyl groups unionized, yet upon coordination to Fe2+, one of its protons is transferred to a second-shell histidine. EPR spectroscopy of NO adducts suggests that both L-DOPA and NO can bind simultaneously to Fe2+ in the active site. Based on these experimental data, a cluster model of the Michaelis complex was constructed and used in DFT investigations of the key steps of the enzymatic reaction, which reproduce the observed reaction regiospecificity and provide clues on how this regiospecificity is achieved. Comparison of the BvDODAα1-L-DOPA complex to other experimental structures of ES complexes of extradiol dioxygenases highlights the catalytic role of the distal “proton-shuttle” histidine.