Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase

Abstract

Copper radical oxidases (CROs) from Auxiliary Activity Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Δloop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

Figure 1

Primary and tertiary structural analyses of the distinct loop region of CgrAAO in relation to 623 AA5_2 catalytic modules. (A) SSN of 623 catalytic modules from the AA5_2 subfamily at a bitscore threshold of 550. Predicted native signal peptides and additional N-terminal modules were removed prior to pairwise sequence alignment. Each node is colored according to the number of amino acids between the two cysteines forming a disulfide bond at the base of the loop. AA5 members with available biochemical data are indicated as galactose oxidases (FgrGalOx,⁻FoxGalOx,FsaGalOx,FauGalOx,ExeGalOx, MreGalOx, FoxGalOxB, and PfeGalOx), alcohol oxidases (CgrAlcOx, CglAlcOx,ChiAlcOx, PorAlcOx,GciAlcOx, AsyAlcOx, AflAlcOx, and FoxAlcOx), AA5_2 oxidases ((PruAlcOx/PruAA5_2A)^,), raffinose oxidases (CgrRafOx,PhuRafOx, and UmaRafOx), and aryl alcohol oxidases (CgrAAO,FoxAAO, and FgrAAO). (B) Multiple sequence alignment of residues forming CgrAAO-extended loops in characterized AA5_2s. (C) Top view of cartoon representation and (D) top view of surface representation of the CgrAAO-WT disulfide bridge and extended loop. For each panel, the copper atom is shown as an orange sphere; cysteine residues C553 and C565 forming the disulfide bridge and active-site residues Cys272, Tyr316, Tyr533, His534, and His632 involved in copper coordination and catalytic activity are depicted as green sticks and light cyan sticks, respectively; C atoms forming the extended loop are in hot pink, and other C atoms are in wheat.

Figure 2

Amino acids of the CgrAAO extended loop in hydrogen bonding with extended galacto-oligosaccharides. Molecular docking studies of raffinose (A), melibiose (B), and lactose (C) in CgrAAO-WT (PDB ID 6RYV) were conducted using AutoDock Vina, as implemented in Chimera. For all panels, the copper atom is depicted as an orange sphere; residues belonging to the extended loop (Trp554 and Gln556) and forming hydrogen bonds with the substrate are depicted as magenta sticks, with hydrogen-bonding moieties in purple/white and corresponding distances in yellow.

Figure 3

Specific activity of CgrAAO-WT and CgrAAO-Δloop on a diversity of potential substrates. Measurements were performed in triplicate at 25 °C in 100 mM sodium phosphate buffer (pH 7) using the horseradish peroxidase (HRP)/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Activities were monitored using 300 mM for carbohydrates, polyols, and diols; 50 mM for methylglyoxal; and 5 mM for aryl alcohols and furans. Reactions were started with the addition of 1.3–65 μmol of purified enzyme.

Figure 4

CgrAAO-WT and CgrAAO-Δloop galactose oxidation product analysis. ¹³C NMR spectra (600 MHz); (A) control reaction showing no conversion of galactose by HRP and catalase, (B) oxidation of d-galactose by CgrAAO-WT showing conversion to the C6 gem-diol, (C) oxidation of d-galactose by CgrAAO-Δloop showing conversion to galactonic acid, (D) d-galacto-1,5-lactone dissolved in 100 mM sodium phosphate buffer, pH 7, showing its equilibrium with d-galactonic acid, and (E) d-galacto-1,5-lactone dissolved in D₂O. All reaction profiles were taken after 24 h incubation with 20 mg of substrate in the presence of catalase and HRP. Carbon signals in purple correspond to the C6-oxidized galactose product, while the red carbon signals correspond to galactonic acid and the blue signals correspond to d-galacto-1,5-lactone. Signals are referenced to the acetone peak at 30.89 ppm (not shown) that was used as an internal standard.

Figure 5

Summary of the observed major products from the oxidation of mono- and oligosaccharides by CgrAAO-WT and CgrAAO-Δloop. *n.a., not assessed; Gal, galactose; Glu, glucose; n.r., nonreducing end; r., reducing end.