Biochemical and structural insights of a recombinant AA16 LPMO from the marine and sponge-symbiont Peniophora sp

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Cairo, Joa o Paulo L. Franco ; Almeida, Dnane V. ; Andrade, Viviane B. ; Telfer, Abbey ; Goncalves, Thiago A. ; Diaz, Daniel E. ; Figueiredo, Fernanda L. ; Brenelli, Livia B. ; Walton, Paul H. ; Garcia, Wanius ; Squina, Fabio M.

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 280, p. 11-pg., 2024-09-18.

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize polysaccharides, leading to their cleavage. LPMOs are classified into eight CAZy families (AA9-11, AA13-17), with the functionality of AA16 being poorly characterized. This study presents biochemical and structural data for an AA16 LPMO (PnAA16) from the marine sponge symbiont Peniophora sp. Phylogenetic analysis revealed that PnAA16 clusters separately from previously characterized AA16s. However, the structural modelling of PnAA16 showed the characteristic immunoglobulin-like fold of LPMOs, with a conserved his-brace motif coordinating a copper ion. The copper-bound PnAA16 showed greater thermal stability than its apo-form, highlighting copper's role in enzyme stability. Functionally, PnAA16 demonstrated oxidase activity, producing 5 mu M H2O2 after 30 min, but showed 20 times lower peroxidase activity (0.27 U/g) compared to a fungal AA9. Specific activity assays indicated that PnAA16 acts only on cellohexaose, generating native celloligosaccharides (C3 to C5) and oxidized products with regioselective oxidation at C1 and C4 positions. Finally, PnAA16 boosted the activity of a cellulolytic cocktail for cellulose saccharification in the presence of ascorbic acid, hydrogen peroxide, or both. In conclusion, the present work provides insights into the AA16 family, expanding the understanding of their structural and functional relationships and biotechnological potential. (AU)

• FAPESP's process: 20/05784-3 - EMU approved in grant 15 / 50590-4: chromatographic system and detectors for analysis of sugars and lignocellulosic monolignols
• Grantee: Fábio Márcio Squina
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 22/08958-8 - Development of microorganisms and thermophilic enzymes involved in the degradation and upcycling of fossil plastics
• Grantee: Fábio Márcio Squina
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/13500-5 - Periplasmic expression, purification and characterization of the inorganic biochemistry of an oxidative enzyme of the insect Bombyx mori (silkworm)
• Grantee: Dnane Vieira Almeida
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 15/50590-4 - Lignin valorization in cellulosic ethanol plants: biocatalytic conversion via ferulic acid to high value chemicals
• Grantee: Fábio Márcio Squina
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 22/05731-2 - BEYOND: establishing a fungal cell factory for recombinant protein production
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• FAPESP's process: 17/17275-3 - Studies of the mode of action of two lytic polysaccharide monooxygenases from insect (order: isoptera): molecular structure, bioinorganic chemistry and biotechnological applications
• Grantee: Wanius José Garcia da Silva
• Support Opportunities: Regular Research Grants