Expansion of Auxiliary Activity Family 5 sequence space via biochemical characterization of six new copper radical oxidases

Abstract

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.

Keywords: Auxiliary Activity Family 5; HMF oxidase; alcohol oxidase; carbohydrate-active enzyme; copper radical oxidase; galactose oxidase.

Fig 1

Specific activities of AA5_2 members on representative compounds. Individual values are presented in Table S2. Specific activity was determined using the HRP-ABTS-coupled assay with 50 mM sodium phosphate buffer, pH 7.0, at room temperature, except for NexGalOx (50 mM sodium acetate buffer, pH 5.0). Measurements were performed in triplicate with 300 mM carbohydrates (galactose, lactose, melibiose, and raffinose), 300 mM glycerol, 10 mM aromatic alcohols [benzyl alcohol and 5-hydroxymethylfurfural (HMF)], and 10 mM aldehydes (methyl glyoxal and glyoxal). Nha, Nectria haematococca; Efe, Epichloe festucae; Nex, Niesslia exilis; Ast, Acremonium strictum; Bsp, Bisporella sp.; and Xpa, Xanthoria parietina.

Fig 2

Tertiary structural comparison of AA5 CRO active sites. Structural models of (A) XpaGalOx, (B) BspGalOx, (C) EfeGalOx, (D) NexGalOx, (E) NhaGalOx, and (F) AstAAO were generated with AlphaFold3 (73) and superposed with an experimental structure of FgrGalOx [PDB 1gof (19), green amino acids], into which galactose was docked previously (40). Conserved residues within respective AA5_2 members from this work are shown as gray sticks with black labels, while non-conserved residues are displayed as blue sticks and labels. Galactose and copper are shown as cyan sticks and orange spheres, respectively. In all cases, AlphaFold did not generate the corresponding active-site Cys-Tyr crosslink (19), but modeled these as two independent sidechains; this did not affect the overall analysis.