L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

Soares, D. M. M.; Goncalves, L. C. P.; Machado, C. O.; Esteves, L. C.; Stevani, C. V.; Oliveira, C. C.; Dorr, F. A.; Pinto, E.; Adachi, F. M. M.; Hotta, C. T.; Bastos, E. L.

doi:10.1101/2020.08.03.235077

bioRxiv · 10.1101/2020.08.03.235077

L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

Soares, D. M. M.·Goncalves, L. C. P.·Machado, C. O.·Esteves, L. C.·Stevani, C. V.·Oliveira, C. C.·Dorr, F. A.·Pinto, E.·Adachi, F. M. M.·Hotta, C. T.·Bastos, E. L.

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Abstract

O_SCPLOWLC_SCPLOW-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 O_SCPLOWLC_SCPLOW-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 O_SCPLOWLC_SCPLOW-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess O_SCPLOWLC_SCPLOW-DOPA, both the betaxanthin and hygroaurin adducts of O_SCPLOWLC_SCPLOW-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other O_SCPLOWLC_SCPLOW-DOPA dioxygenases. The rate constants for the conversion of O_SCPLOWLC_SCPLOW-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.

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Soares, D. M. M., Goncalves, L. C. P., Machado, C. O., Esteves, L. C., Stevani, C. V., Oliveira, C. C., Dorr, F. A., Pinto, E., Adachi, F. M. M., Hotta, C. T., Bastos, E. L.. 2020-08-04. L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production. https://doi.org/10.1101/2020.08.03.235077

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