Beta vulgaris is the main commercial source of betalain pigments, which are used as food colorants and dietary supplements among other applications (1). The key step in betalains biosynthesis is oxidative ring opening of L-3,4-dihydroxyphenylalanine (L-DOPA) catalysed by L-DOPA extradiol dioxygenase (DODA, EC: 1.13.11.29), which yields 4,5-seco-DOPA (2). This product is metastable and undergoes cyclization leading to the bioactive core of all betalains - betalamic acid. DODA enzymes from Escherichia coli (3), Amanita muscaria (4) and Gluconacetobacter diazotrophicus (5), besides the 4,5-extradiol activity, also cleave the aromatic ring of L-DOPA at the C2-C3 bond, which yields 2,3-seco-DOPA and finally, muscaflavin. It is currently unknown what is the origin of this dual activity of these enzymes.
With the use of X-ray macromolecular crystallography we aimed to shed light on the significance of residues in the substrate binding cavity on the reaction stereospecificity. We pointed the residues suspected to play an important role in substrate positioning to subsequently subject these residues to mutagenesis to confirm their importance in the substrate binding and reaction outcome. As an iron substitute we used nickel, manganese and cobalt ions and we have examined the substitution impact on the active site arrangement. We present structural details of BvDODA complexes with inactive substrate analogs (4-nitrocatechol, 4-nitro-L-DOPA, mimosine) as well as BvDODA complexes with Fe-substitutes.