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

Author(s):Show less -

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.

• Document type: Journal article
• Source: ACS OMEGA; v. 7, n. 18, p. 10-pg., 2022-05-10.

Abstract

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