PT  - JOURNAL ARTICLE
AU  - Soares, Douglas M. M.
AU  - Gonçalves, Letícia C. P.
AU  - Machado, Caroline O.
AU  - Esteves, Larissa Cerrato
AU  - Stevani, Cassius V.
AU  - Oliveira, Carla C.
AU  - Dörr, Felipe A.
AU  - Pinto, Ernani
AU  - Adachi, Flávia M. M.
AU  - Hotta, Carlos T.
AU  - Bastos, Erick L.
TI  - L-DOPA dioxygenase of the fly agaric toadstool: revision of the <em>dodA</em> gene sequence and mechanism of enzymatic pigment production
AID  - 10.1101/2020.08.03.235077
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.03.235077
4099  - http://biorxiv.org/content/early/2020/08/04/2020.08.03.235077.short
4100  - http://biorxiv.org/content/early/2020/08/04/2020.08.03.235077.full
AB  - l-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous l-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of l-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess l-DOPA, both the betaxanthin and hygroaurin adducts of l-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other l-DOPA dioxygenases. The rate constants for the conversion of l-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.Competing Interest StatementThe authors have declared no competing interest.