Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity

Abstract

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5\_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5\_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5\_2 enzymes towards galactose-containing saccharides.

We are grateful to Pom Geslin-Vinck for her assistance in constructing some of the CgrAlcOx loop variants. This study was supported by (a) the ‘Centre National de la Recherche Scientifique’ (CNRS), (b) the French ‘Agence Nationale de la Recherche’ (ANR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) through the joint ‘Projet de Recherche Collaboratif International’/‘Strategic Partnership Grants for Projects’ program, supporting the project entitled ‘FUNTASTIC—Fungal copper radical oxidases as new biocatalysts for the valorization of biomass carbohydrates and alcohols’ (ANR-17-CE07-0047, NSERC STPGP 493781–16) and (c) the Spanish Ministry of Innovation and Sciences (PID2019-106370RB-I00).

Peer Reviewed

Postprint (published version)