Auxiliary Activity Family 5

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• Author: ^^^Maria Cleveland^^^ and ^^^Yann Mathieu^^^
• Responsible Curator: ^^^Harry Brumer^^^

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Auxiliary Activity Family AA5
Fold	Seven-bladed β-propeller
Mechanism	Copper Radical Oxidase
Active site residues	known
CAZy DB link
http://www.cazy.org/AA5.html

General Properties

Enzymes from the CAZy family AA5 are mononuclear copper-radical oxidases (CRO) that perform catalysis independently of 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 1.2.3.15) [1] and subfamily AA5_2 contains galactose oxidases (EC 1.1.3.9) [2], as well as the more recently discovered raffinose oxidases [3, 4], aliphatic alcohol oxidases (EC 1.1.3.13) [4, 5, 6] and aryl alcohol oxidase (EC 1.1.3.7) [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]. While this first report already established FgrGalOx as a metalloenzyme; its copper requirement was later confirmed [12]. Until 2015 the 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 [13]. Furthermore, an AA5 enzyme (GlxA) has been shown to have activity on glycolaldehyde and the deletion mutant of GlxA from Streptomyces lividans showed loss of glycan accumulation at hyphal tips [14].

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 [15], the AA5_2 members accept alcohol containing substrates and oxidatively form the corresponding aldehyde or acid [16, 17].

AA5_1

The AA5_1 enzymes have been characterized as glyoxal oxidases (EC 1.2.3.15) and show activity on simple aldehydes, α-hydroxycarbonyl, and α-dicarbonyl compounds, with the highest activity observe don glyoxal, methyl glyoxal and glycolaldehyde [9, 15, 18, 19, 20]. Most characterized AA5_1 enzymes show the highest activity on methyl glyoxal [18, 20, 21], however, two glyoxal oxidases form Pycnoporus cinnabarinus demonstrate the highest catalytic efficiency on glyoxylic acid as the substrate [22].

AA5_2

The founding member of AA5_2 from Fusarium graminearum is a galactose oxidases (FgrGalOx), which catalyzes the regioselective oxidation of the C6-hydroxyl group on the monosaccharide galactose (EC 1.3.3.7) [23]. The range of substrates oxidized by FgrGalOx includes galactose derivatives such as 1-methyl-b-galactopyranoside [24], and galactose-containing oligo-and polysaccharides including: lactose, melibiose, raffinose, galactoxyloglucan, galactomannan and galactoglucomannan [17, 25]. Most other AA5_2s from the Fusarium family, such as F. oxysporum [24, 26, 27, 27, 27, 27, 28, 28, 29, 30, 31, 32] 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]. Due to these enzymes exhibiting high specificity towards 1-butanol, benzyl alcohol and cinnamyl alcohol, they were classified as general alcohol oxidases (EC 1.3.3.13) [5]. In addition, two alcohol oxidases were characterized from the rice blast pathogen Pyricularia oryzae (PorAlcOx) and from C. 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 1.1.3.7 and EC 1.1.3.47) [7]. In addition, two AA5_2 homologs from Fusarium species have been classified also as aryl alcohols oxidases (FgrAAO and FoxAAO) [8] and 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 [33, 34, 35, 36, 37, 38, 39, 40] while the ability to oxidize aromatic alcohols to the corresponding aldehydes can be utilized in a variety of applications within the food and fragrance industry [41]. Similarly, the ability of CROs to convert HMF into the bi-functional polymer precursors DFF and FDCA, which is important in the context of bio-polymer manufacturing [42, 43].

Kinetics and Mechanism

Figure X. 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 mediated through the presence of a redox active Tyr-Cys cofactor. In AA5_2 the Tyr-Cys cofactor exhibits an unusually low reduction potential (+275 mV) [44, 45, 46] compared to unmodified tyrosine in solution (> +800 mV) or in other enzymatic systems [47]. 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, 48, 49]. In contrast, AA5_1 have reduction potential of around +640 mV [18] which could explain the different oxidizing power of the two subfamilies [15, 46]. 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 [15, 46]. In the archetypal AA5_2 member, FgrGalOx, the Trp290His substitution increased the redox potential of the resulting enzyme from +400 mV to +730 mV [50]; however, it also decreased the catalytic efficiency by 1000-fold [51] and perturbed the stability of the [Cu2+ Tyr·] metallo-radical complex at neutral pH [52]. CgrAlcOx and CgrAAO have been speculated to have a lower redox potential than FgrGalOx due to the secondary amino acid substitution (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, 15, 18, 51, 53, 54, 55, 56], including some theoretical and biomimetic models possessing mechanistic similarities [57, 58]. 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)[59, 60, 61, 62, 63].

Catalytic Residues

Figure X. 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, 18, 53, 56]. Based on the copper coordination environment, AA5 proteins are type 2 “non-blue” copper category due to the nitrogen and oxygen ligands [64]. The unique feature of the AA5 is the covalently linked equatorial tyrosine with an adjacent cysteine by a thioether bond (Tyr 272 and Cys 228 in the archetypal FgrGalOx) [64, 65]. 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 [66].

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, radial stability and redox activity by hydrogen bonding, delocalization and/or by protecting the thioether bond from solvent [1, 2, 15, 49]. This residue in AA5_1, based on sequence alignments, has been conserved as a histidine [15], while characterized AA5_2s enzymes have an aromatic residue at this position: a tryptophan in galactose oxidases (W290 in FgrGalOx) [4, 49], a phenylalanine in the Colletotrichum aliphatic alcohol oxidases [5], and a tyrosine in the raffinose oxidases [3, 4] and aryl alcohol oxidase from Colletotrichum graminicola [3, 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 [14, 67].

Three-dimensional Structures

Figure X. 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].

AA5 share a seven-bladed β-propeller fold [5, 7, 64] as 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 [64]. Other characterized AA5_2 enzymes from Fusarium species contain CBM32 [4, 24, 26, 68], even though some do not display canonical galactose oxidase activity (ex. FgrAAO and FoxAAO) [4, 8]. In contrast, CgrAlcOx, CglAlcOx and ChiAlcOx do not poses 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 domains involvement in enzyme anchoring on the plant surface [6]. In addition, the fusion of a galactose oxidase with a CBM29 has shown an increase in catalytic efficiency of the construct on galactose-containing hemicelluloses compared to WT [69].

Family Firsts

First stereochemistry determination

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First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

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