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Auxiliary Activity Family 5

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Auxiliary Activity Family AA5
Fold Seven-bladed β-propeller
Mechanism Copper Radical Oxidase
Active site residues known
CAZy DB link

General Properties

Enzymes from the CAZy family Auxiliary Activity 5 (AA5) are mononuclear copper-radical oxidases (CRO) that perform catalysis without complex organic cofactors such as FAD or NADP and use oxygen as their electron acceptor (EC 1.1.3.-). Family AA5 enzymes are classified in two subfamilies: subfamily AA5_1 contains characterized glyoxal oxidases (EC [1] and subfamily AA5_2 contains characterized galactose oxidases (EC [2], as well as the more recently discovered raffinose oxidases [3, 4], aliphatic alcohol oxidases (EC [4, 5, 6] and aryl alcohol oxidase (EC [7, 8].

The most studied enzyme in subfamily AA5_1 is the glyoxal oxidase from Phanerochaete chrysosporium discovered in 1987 [9]. For subfamily AA5_2, the archetypal galactose-6 oxidase from Fusarium graminearum (FgrGalOx) was first reported in 1959 from cultures of Polyporus circinatus (later renamed Fusarium graminearum [10, 11]. Until 2015, characterized enzymes from the AA5_2 subfamily were found to exhibit mainly galactose oxidase activity, but since then novel non-carbohydrate oxidase enzymes were found [4, 5, 6, 7, 8].

An AA5 enzyme from Arabidopsis thaliana, whose sequence does not fall within the two known subfamilies, has been identified as a putative galactose oxidase, RUBY, and has been demonstrated to promote the cell-to-cell adhesion in the seed coat epidermis of Arabidopsis [12]. Furthermore, an enzyme distantly related to AA5 called GlxA, has been shown to have activity on glycolaldehyde and the GlxA deletion mutant from Streptomyces lividans showed a loss of glycan accumulation at hyphal tips [13].

Substrate Specificities

An important distinction between the AA5_1 and AA5_2 subfamilies, is that while AA5_1 enzyme catalyze the two-electron oxidation of simple aldehydes and hydroxycarbonyl [14], the AA5_2 members accept alcohol containing substrates and usually oxidatively form the corresponding aldehyde and in some instances can also oxidize the aldehyde into the corresponding acid [15, 16].


The AA5_1 enzymes have been characterized as glyoxal oxidases (EC and show activity on simple aldehydes, α-hydroxycarbonyl, and α-dicarbonyl compounds, with the highest activity observed on glyoxal, methyl glyoxal and glycolaldehyde [9, 14, 17, 18, 19]. In contrast, two glyoxal oxidases form Pycnoporus cinnabarinus demonstrate the highest catalytic efficiency on glyoxylic acid as the substrate [20].


The founding member of AA5_2 from Fusarium graminearum is a galactose oxidase (FgrGalOx), which catalyzes the regioselective oxidation of the C6-hydroxyl group on the monosaccharide galactose (EC [21]. The range of substrates oxidized by FgrGalOx also includes galactose derivatives such as 1-methyl-b-galactopyranoside [22], and galactose-containing oligo-and polysaccharides including: lactose, melibiose, raffinose, galactoxyloglucan, galactomannan and galactoglucomannan [16, 23]. Most other AA5_2s from the Fusarium family, such as F. oxysporum [24], F. sambucinum [22], and F. acuminatum [25] have similar substrate specificities to FgrGalOx.

In 2015, two AA5_2 oxidases with distinct substrate profiles were discovered from Colletotrichum graminicola and Colletotrichum gloeosporioides (CgrAlcOx and CglAlcOx) that were essentially inactive on galactose and galactosides, but efficiently oxidized the primary hydroxyl of diverse aliphatic and aromatic alcohols [5]. Since these enzymes exhibited high specificity towards 1-butanol, benzyl alcohol and cinnamyl alcohol, they were classified as general alcohol oxidases (EC [5]. In addition, two alcohol oxidases were characterized from the rice pathogen Pyricularia oryzae (PorAlcOx) and from Colletotrichum higginsianum (ChiAlcOx) with both enzymes showing prominent activity on n-butanol, ethanol, 1,3-butanediol and glycerol [6]. Since then, more AA5_2 enzymes from various fungal origins have been characterized as general alcohol oxidases, some being able to efficiently oxidize both carbohydrate and non-carbohydrate substrates (ex.AflAlcOx) [4].

Furthermore, another AA5_2 member from C. graminicola has been characterized as an aryl-alcohol oxidase (CgrAAO) due to its high specific activity towards substituted benzyl alcohols and 5-hydroxymethylfurfural (HMF) (EC and EC [7]. In addition, two AA5_2 homologs from Fusarium species have been also classified as aryl alcohols oxidases (FgrAAO and FoxAAO) [8] while other AA5_2 members have been shown to have prominent activity on HMF with enzymes being able to fully oxidize the furan substrate to FDCA (GciAlcOx) [4].

The last distinct group of AA5_2 enzymes based on substrate specificity are the raffinose-specific oxidases (EC 1.3.3.-). These enzymes show only negligible activity on galactose, were not active towards aliphatic alcohols, however, they showed the highest activity for the tri-saccharide raffinose, the diol glycerol, and the glycolaldehyde dimer [3, 4].

Hence, the AA5_2 family contains a diverse set of enzymes with galactose oxidases, raffinose oxidases, general alcohol oxidases (carbohydrate and non-carbohydrate) and aryl alcohol oxidases.

The ability of CROs to oxidize galactose-containing sugars can be utilized for the production of biomaterials from biomass sources [26, 27, 28, 29, 30, 31, 32, 33] while their ability to oxidize linear and aromatic alcohols to the corresponding aldehydes can be utilized in a variety of applications within the food and fragrance industry [34]. Similarly, the ability of CROs to convert HMF into the bi-functional polymer precursors DFF and FDCA can be potentially utilized in the context of bio-polymer manufacturing [35, 36].

Kinetics and Mechanism

Figure 1. Reaction mechanism of copper radical oxidases. A. First half-reaction – oxidation of substrate. B. Second-half reaction – regeneration of active site radical. PT – proton transfer, HAT – hydrogen atom transfer, ET – electron transfer. This figure is adapted from [5].

AA5 enzymes oxidize their substrate with concurrent reduction of oxygen to hydrogen peroxide in a two-electron process mediated through a free-radical-coupled copper complex via the presence of a redox active Tyr-Cys cofactor. In AA5_2 the Tyr-Cys cofactor exhibits an unusually low reduction potential (+275 mV) [37, 38, 39] compared to unmodified tyrosine in solution (> +800 mV) or in other enzymatic systems [40]. Several factors could contribute to this phenomenon by increasing the stability of the protein free radical including π-stacking with aromatic residues and the electron donating effect of the thioether linkage [2, 41, 42]. In contrast, AA5_1 have a reduction potential around +640 mV [17] which could explain the different oxidizing power of these two subfamilies [14, 39]. One reason for the higher reduction potential of glyoxal oxidases could be subtitution of the secondary shell amino acid Trp in AA5_2 with a His in AA5_1 [14, 39]. In the archetypal AA5_2 member, FgrGalOx, the Trp290His substitution increased the reduction potential of the resulting enzyme from +400 mV to +730 mV [43]; however, it also decreased the catalytic efficiency by 1000-fold [44] and affected the stability of the [Cu2+ Tyr·] metallo-radical complex at neutral pH [45]. CgrAlcOx and CgrAAO have been speculated to have a lower reduction potential than FgrGalOx due to their secondary shell amino acid substitutions (Phe in CgrAlcOx and Tyr in CgrAAO) [5, 7].

A substantial amount of spectroscopic evidence has supported a ping-pong mechanism for AA5 enzymes [2, 14, 17, 44, 46, 47, 48, 49], including some theoretical and biomimetic models possessing mechanistic similarities [50, 51]. The reaction progresses through a two-step process: the first half-reaction performs the oxidation of the substrate, while the second half-reaction regenerates the oxidation state of the active-site copper with concurrent reduction of molecular oxygen to hydrogen peroxide. Each half reaction consists of three steps: proton transfer (PT), hydrogen atom transfer (HAT), and an electron transfer (ET).

Other work, focused on optimizing the industrial potential of AA5 enzymes, has shown that AA5_2 enzymes have increased activity with the addition of peroxidases (horse-radish peroxidase and catalase) and small molecule activators (potassium ferricyanide and magnesium (III) acetate) [52, 53, 54, 55, 56].

Catalytic Residues

Figure 2. Active site residues of copper radical oxidases FgrGalOx, Cu ion in orange (PDB ID 1GOF).

The redox-active center of AA5 oxidases comprises a copper ion that is stabilized by two tyrosines and two histidines (in FgrGalOx, these are Tyr272, Tyr495, His496, His581), resulting in a distorted square pyramidal geometry [2, 17, 46, 49]. Based on the copper coordination environment, AA5 proteins are type 2 “non-blue” copper category due to the nitrogen and oxygen ligands [57]. The unique feature of AA5 enzymes is the covalently linked equatorial tyrosine with an adjacent cysteine by a thioether bond (Tyr 272 and Cys 228 in the archetypal FgrGalOx) [57, 58]. The thioether linkage forms spontaneously in the presence of copper and has been shown to stabilize the radical though delocalization that forms on the equatorial tyrosine during catalysis [59].

Another important feature of AA5 enzymes is a secondary shell amino acid that is located on top of the tyrosine-cysteine cofactor. It has been speculated to be critical in determining the substrate specificity, radical stability and redox activity by hydrogen bonding, delocalization and/or by protecting the thioether bond from solvent [1, 2, 14, 42]. This residue in AA5_1, based on sequence alignments, has been conserved as a histidine [14], while characterized AA5_2 enzymes have an aromatic residue at this position: a tryptophan in galactose oxidases (W290 in FgrGalOx) [4, 42], a phenylalanine in the Colletotrichum aliphatic alcohol oxidases [5], whereas a tyrosine is present in the raffinose oxidases [3, 4] and aryl alcohol oxidase from Colletotrichum graminicola [7]. Furthermore, an AA5 enzyme from Streptomyces lividans with activity on glycolaldehyde possesses a tryptophan as the stacking secondary shell residue whose indole ring is oriented differently compared to FgrGalOx, which may affect the substrate specificity [13, 60].

Three-dimensional Structures

Figure 3. Crystal structure of copper radical oxidases. A. FgrGalOx (PDB ID 1GOF), Copper ion in orange and B. CgrAlcOx (PDB ID 5C86), Copper ion in grey. This figure is adapted from [5].

AA5s share a seven-bladed β-propeller fold [5, 7, 57] for the catalytic domain containing the active site. The archetypal FgrGalOx contains three domains: domain 1 has a “β sandwich” structure identified as a carbohydrate binding module (CBM32) with affinity for galactose, domain 2 is the catalytic domain and domain 3 is the smallest, which forms a hydrogen bonding network to stabilize domain 2 [57]. Other characterized AA5_2 enzymes from Fusarium species contain CBM32 [4, 22, 24, 61], even though some do not display canonical galactose oxidase activity (ex. FgrAAO and FoxAAO) [4, 8]. In contrast, CgrAlcOx, CglAlcOx and ChiAlcOx do not possess any CBM [5, 6], while CgrAAO and CgrRafOx have a PAN domain present instead [3, 7]. PorAlcOx contained a WSC domain that was able to bind xylans and fungal chitin/β-1,3-glucan, implicating the domain's involvement in enzyme anchoring on the plant surface [6]. In addition, the fusion of a galactose oxidase with a CBM29 has shown to increase the catalytic efficiency of the construct on galactose-containing hemicelluloses compared to WT [62].

Family Firsts

First AA5_1 enzyme discovered
The glyoxal oxidase from Phanerochaete chrysosporium discovered in 1987 [9].
First AA5_2 enzyme discovered
The archetypal galactose-6 oxidase from Fusarium graminearum (FgrGalOx) discovered in 1959 [11].
Copper requirement confirmed
While this first report already established FgrGalOx as a metalloenzyme; its copper requirement was later confirmed [63].
First 3-D structure
The first crystallography structure of AA5 was of the archetypal FgrGalOx in 1991 [58].


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