Structure-function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

Abstract

Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure-function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications.

Figure 1. Abbreviated mechanism of galactose oxidase.

(a) The first half-reaction in which the enzyme is reduced while oxidizing the alcohol to the aldehyde. (b) The second half-reaction reduces O₂ to H₂O₂ while oxidizing copper¹⁺ to the resting +2 oxidative state (reviewed in ref. ; see ref. for a detailed mechanism). ET, electron transfer; PT, proton transfer.

Figure 2. Phylogeny of AA5.

Subfamilies 1 (AA5_1) and 2 (AA5_2) are indicated. GenBank identifiers (UniProt identifier Q01745 in the case of the F. graminarium GalOx) are given for all sequences available in the public release of the CAZy Database as of June 2015; full genus and species names of source organisms can be obtained from the respective GenBank entries. AA5 members for which enzymological data have been conclusively linked to protein sequence are indicated as (methyl)glyoxal oxidases (GlyoxOx), galactose oxidases (GalOx; refs , , , , and references therein), or general alcohol oxidases (AlcOx; this study).

Figure 3. Initial activity screen of CgrAlcOx against carbohydrates.

Saccharides and glycerol were assayed at 50 mM and xyloglucan was assayed at 1 g l⁻¹. Results shown are averages of duplicate measurements from a single experimental replicate; error bars represent s.d.'s.

Figure 4. 3D structure of CgrAlcOx.

(a) At the N terminus (colour-ramped blue to yellow), seven Kelch motifs enclose the copper-binding site in a β-propeller arrangement, while the C terminus displays a nine-stranded β-barrel (orange to red). (b) Orthogonal view. The Cu ion is showed as a grey-shaded sphere. (c) Overlap of CgrAlcOx (dark) and FgrGalOx (pale, 2EIE), in divergent (‘wall-eyed') stereo highlighting the additional N-terminal CBM32 domain of the latter (pale pink).

Figure 5. Active site of CgrAlcOx.

(a) Observed electron density map for the active site, maximum likelihood (REFMAC) weighted 2F_obs–F_calc map contoured at 0.59 ųDa⁻¹, with the anomalous difference density map (red, λ=0.979 Å) contoured at 2σ. Copper is coordinated by residues Y120, Y334, H335 and H423 in the centre of the N-terminal β-propeller. (b) X-band EPR spectrum (9.3 GHz, 155 K) of Cu-CgrAlcOx in 10% v/v glycerol (black) with simulation (red). The spectrum of CglAlcOx was identical (Supplementary Fig. 12). (c) Divergent stereoscopic view of the overlay of CgrAlcOx (dark) and FgrGalOx (pale) copper-binding centres. F138 in CgrAlcOx is substituted by W290 in FgrGalOx.