Two Fusarium copper radical oxidases with high activity on aryl alcohols

Abstract

Background Biomass valorization has been suggested as a sustainable alternative to petroleum-based energy and commodities. In this context, the copper radical oxidases (CROs) from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts for the selective oxidation of primary alcohols to aldehydes. Originally defined by the archetypal galactose 6-oxidase from Fusarium graminearum, fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of native CROs for applications. However, only 10% of the annotated AA5_2 members have been characterized to date. Results Here, we define two homologues from the filamentous fungi Fusarium graminearum and F. oxysporum as predominant aryl alcohol oxidases (AAOs) through recombinant production in Pichia pastoris, detailed kinetic characterization, and enzyme product analysis. Despite possessing generally similar active-site architectures to the archetypal FgrGalOx, FgrAAO and FoxAAO have weak activity on carbohydrates, but instead efficiently oxidize specific aryl alcohols. Notably, both FgrAAO and FoxAAO oxidize hydroxymethyl furfural (HMF) directly to 5-formyl-2-furoic acid (FFCA), and desymmetrize the bioproduct glycerol to the uncommon L-isomer of glyceraldehyde. Conclusions This work expands understanding of the catalytic diversity of CRO from AA5_2 to include unique representatives from Fusarium species that depart from the well-known galactose 6-oxidase activity of this family. Detailed enzymological analysis highlights the potential biotechnological applications of these orthologs in the production of renewable plastic polymer precursors and other chemicals.

A simple and direct ionic chromatography method to monitor galactose oxidase activity

“…32 Taking advantage of recombinant enzyme production pipelines, a large diversity of AA5_2 enzymes has been recently characterized. 9,10,33–36…”

“…32 Taking advantage of recombinant enzyme production pipelines, a large diversity of AA5_2 enzymes has been recently characterized. 9,10,[33][34][35][36] GalOx activity has historically been measured by indirect, spectrophotometric methods that rely on quantifying the coproduct H 2 O 2 using a coupled horseradish peroxidase (HRP)/ 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. 37 Despite widespread use, such assays have certain limitations due to the cross-reactivity of H 2 O 2 or the instability of the reporter molecule (ABTS cation radical).…”

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

“…HMF, via its oxidized derivatives, is a biomass-derived feedstock of particular contemporary interest and an exemplar aryl alcohol substrate of AA5 CROs. − We previously showed that the dialdehyde DFF is the sole product of HMF oxidation by wild-type Cgr AAO . As the Cgr AAO-Δloop variant gained the ability to oxidize DFF at the expense of HMF activity (Table ), product analysis by NMR experiments was performed on these two substrates.…”

“…1.1.3.13), − and aryl alcohol oxidases (AAO, E.C. 1.1.3.7), , which convert the primary alcohol of their respective substrates to the corresponding aldehyde. Catalysis in AA5 members is mediated by a mononuclear copper center, which is coordinated by a unique cross-linked tyrosine–cysteine radical cofactor; molecular oxygen (O 2 ) serves as the final electron acceptor to generate hydrogen peroxide (H 2 O 2 ) as a co-product. , …”

“…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 ( Fgr GalOx, − Fox GalOx, Fsa GalOx, Fau GalOx, Exe GalOx, Mre GalOx, Fox GalOxB, and Pfe GalOx), alcohol oxidases ( Cgr AlcOx, Cgl AlcOx, Chi AlcOx, Por AlcOx, Gci AlcOx, Asy AlcOx, Afl AlcOx, and Fox AlcOx), AA5_2 oxidases (( Pru AlcOx/ Pru AA5_2A) , ), raffinose oxidases ( Cgr RafOx, Phu RafOx, and Uma RafOx), and aryl alcohol oxidases ( Cgr AAO, Fox AAO, and Fgr AAO). (B) Multiple sequence alignment of residues forming Cgr AAO-extended loops in characterized AA5_2s.…”