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Reannotation of Fly Amanita L-DOPA Dioxygenase Gene Enables Its Cloning and Heterologous Expression

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Soares, Douglas M. M. ; Goncalves, Leticia C. P. ; Machado, Caroline O. ; Esteves, Larissa C. ; Stevani, Cassius, V ; Oliveira, Carla C. ; Dorr, Felipe A. ; Pinto, Ernani ; Adachi, Flavia M. M. ; Hotta, Carlos T. ; Bastos, Erick L.
Total Authors: 11
Document type: Journal article
Source: ACS OMEGA; v. 7, n. 18, p. 10-pg., 2022-05-10.

The L-DOPA dioxygenase of Amanita muscaria (AmDODA) participates in the biosynthesis of betalain-and hygroaurin-type natural pigments. AmDODA is encoded by the dodA gene, whose DNA sequence was inferred from cDNA and gDNA libraries almost 30 years ago. However, reports on its heterologous expression rely on either the original 5'-truncated cDNA plasmid or artificial gene synthesis. We provide unequivocal evidence that the heterologous expression of AmDODA from A. muscaria specimens is not possible by using the coding sequence previously inferred for dodA. Here, we rectify and reannotate the full-length coding sequence for AmDODA and express a 205-aa His-tagged active enzyme, which was used to produce the L-DOPA hygroaurin, a rare fungal pigment. Moreover, AmDODA and other isozymes from bacteria were submitted to de novo folding using deep learning algorithms, and their putative active sites were inferred and compared. The wide catalytic pocket of AmDODA and the presence of the His-His-His and His-His-Asp motifs can provide insight into the dual cleavage of L-DOPA at positions 2,3 and 4,5 as per the mechanism proposed for nonheme dioxygenases. (AU)

FAPESP's process: 19/12605-0 - Bioluminescence of Fungi, Millipede and Marine Organisms: Chemical and Biological Aspects
Grantee:Douglas Moraes Mendel Soares
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 19/15412-9 - Conjugates of flower pigments and silica: smart and biocompatible hybrid materials
Grantee:Erick Leite Bastos
Support Opportunities: Regular Research Grants
FAPESP's process: 17/22501-2 - Electronic chemiexcitation in biological systems: bioluminescence and photochemistry in the dark
Grantee:Etelvino José Henriques Bechara
Support Opportunities: Research Projects - Thematic Grants
FAPESP's process: 18/25842-8 - Sources, Targets and Biological Responses of Triplet Species and Singlet Oxygen: Pathogenicity and Quenching.
Grantee:Leticia Christina Pires Gonçalves
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 15/24760-0 - Effect of antioxidant plant pigments on the viability of cells subjected to oxidative photoinduced stress
Grantee:Caroline de Oliveira Machado
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 13/07914-8 - FoRC - Food Research Center
Grantee:Bernadette Dora Gombossy de Melo Franco
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 19/06391-8 - Photophysical characterization and anti-inflammatory potential of betalains
Grantee:Erick Leite Bastos
Support Opportunities: Regular Research Grants