Auxiliary Activity Family 3

This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.

• Author: ^^^Roland Ludwig^^^ and ^^^Daniel Kracher^^^
• Responsible Curator: ^^^Roland Ludwig^^^

---

Auxiliary Activity Family AA3
Clan	AA3
Mechanism	FAD-dependent substrate oxidation
Active site residues	known
CAZy DB link
http://www.cazy.org/AA3.html

General properties and substrate specificities

Figure 1. Reaction cycle of AA3 flavoenzymes. In the reductive half-reaction substrate oxidation leads to the formation of a two-electron reduced FADH2 (flavohydroquinone). During the oxidative half-reaction the flavin cofactor is re-oxidized via electron transfer to a suitable electron acceptor. Exemplary reactions for each AA3 sub-family are shown.

Enzymes of the CAZY family AA3 are widespread and catalyse the oxidation of alcohols or carbohydrates with the concomitant formation of hydrogen peroxide or hydroquinones [1]. AA3 enzymes are most abundant in wood-degrading fungi where they typically display a high multigenicity [2, 3]. The main function of fungal AA3 enzymes is the stimulation of lignocellulose degradation in cooperation with other AA-enzymes such as peroxidases (AA2) [4] or lytic polysaccharide monooxygenases (AA9) [5, 6]. In yeast, AA3 enzymes are involved in the catabolism of alcohols [7], and some AA3 genes identified in insects are thought to be relevant for immunity and development [8, 9]. Recently, a bacterial AA3 enzyme with unknown biological function was isolated and characterized [10]. The functionally diverse enzymes of family AA3 all belong to the structurally related glucose-methanol-choline (GMC) family of oxidoreductases and require a flavin-adenine dinucleotide (FAD) cofactor for catalytic activity. Based on their sequences members of the AA3 family were divided into four subfamilies in the CAZy database (Figure 1). Family AA3_1 contains the flavodehydrogenase domain of cellobiose dehydrogenase (EC 1.1.99.18), family AA3_2 includes aryl alcohol oxidase (EC 1.1.3.7), glucose oxidase (EC 1.1.3.4), glucose dehydrogenase (EC 1.1.5.9) and pyranose dehydrogenase (EC 1.1.99.29), family AA3_3 consists of alcohol (methanol) oxidases (EC 1.1.3.13) and family AA3_4 comprises pyranose oxidoreductases (EC 1.1.3.10).

Subfamily 1 - CDH

Kinetics and Mechanism

Cellobiose dehydrogenase (CDH, EC 1.1.99.15 cellobiose:quinone oxidoreductase) is a flavohaemoprotein (or flavocytochrome) secreted by lignocellulose degrading, phytopathogenic and saprotrophic fungi belonging to the phyla of Basidiomycota and Ascomycota [11]. An important in vivo function of CDH is the reduction of lytic polysaccharide monooxygenases belonging to the auxiliary activity families AA9 and AA10 [12, 13]. CDHs oxidize a variety of lignocellulose-derived saccharides to their corresponding 1,5-lactones. All CDHs show a high catalytic efficiency towards soluble, β-(1,4)-linked cellulose derived di- oand oligosaccharides like cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose and the structural analogue lactose. Also hemicellulose- and starch- derived oligosaccharides, such as xylo- or manno or maltooligosaccharides are oxidized by a number of CDHs, although with lower catalytic efficiency [11, 14]. CDHs typically discriminate monosaccharides and show a low catalytic efficiency towards glucose, galactose and mannose [11, 15].

CDH has a bipartite structure, which comprises an N-terminal cytochrome domain and a catalytic flavodehydrogenase domain (of the GMC-oxidoreductase family) within a single polypeptide chain. The domains are connected by a papain-sensitive interdomain protein linker which typically comprises 15 - 20 amino acids. The oxidation of carbohydrates occurs at the flavodehydrogenase domain, which binds substrates in a binding (B-) site and thereby orients the anomeric carbon atom of the glucose moiety bound in the cytalytic (C-)site towards the N5 atom of the isoalloxazine ring [16]. This orientation allows oxidative attack of cellobiose by the catalytic histidine by a general hydride transfer mechanism, in which the catalytic His initially abstracts a proton from the C1 hydroxyl group [17]. Transfer of the anomeric (C1) hydrogen to the N5 atom of the FAD cofactor then results in a 2-electron reduced FAD (hydroquinone form), while cellobiose is oxidized to cellobionolactone.

Stopped-flow spectrophotometry showed that mixing of CDH with cellobiose results in rapid reduction of the FAD cofactor in the flavodehydrogenase domain, followed by a slower interdomain electron transfer to the b-type heme in the cytochrome domain [18]. The electron transfer from FAD to heme b was further strengthened by the observed midpoint potentials of the cofactors. The heme b redox potential is always higher than that of FAD, typically between +100 - +150 mV. However, the cytochrome domain is not the only electron acceptor and several substituted quinones, redox dyes and metal complexes have been reported as electron acceptors directly interacting with the FAD [11, 15].

Based on phylogenetic, catalytic and molecular differences CDHs are classified into three classes [19]. Class-I CDHs are of basidiomycetous origin, feature a cellulose binding site in the flavodehydrogenase domain, show a strong discrimination of monosaccharides and have an acidic pH optimum. Class-II CDHs are of ascomycetous origin. Subclass CDH-IIA features a carbohydrate binding module (CBM1), whicle CDH-IIB lacks a CDM or a cellulose binding site. Ascomycetous CDHs show a broader substrate spectrum can have acidic or alkaline pH optima [20]. No member From the phylogenetic group of Class-III CDHs has been characterized yet.

Catalytic Residues

The FAD cofactor in the flavodehydrogenase domain is noncovalently bound. The substrates anomeric carbon atom is oriented towards N5 of the FAD isoalloxazine ring at a distance of approx. 2.9 Å [16] and close to the general base histidine (HXXX in 1KDG, HYYY in 4QM6, ZZZ in 4QM7). Important residues for substrate binding are XXDXX and essential for the interdomain electron transfer is R7XX [21].

Three-dimensional structures

The crystal structure of the isolated cytochrome domain from P. chrysosporium CDH was reported in 2000 at a resolution of 1.9 Å (PDB ID 1D7C, [22]). The fragment folds into a compact globular structure which resembles the antibody fold of the heavy-chain Fab fragment. The propionate arm of the heme b is partly solvent exposed. The heme iron is hexa-coordinated by Met and His as axial ligands [23]. The crystal structure of the P. chrysosporium flavodehydrogenase domain (1.6 Å resolution, 1KDG) was reported in 2002 [24]. The flavin binding domain features the typical, flavin-binding βαβ-motif - the Rossmann-fold. The FAD moiety is non-covalently bound to the enzyme. Full-length structures of Neurospora crassa and Myriococcum thermophilum CDH were reported in 2015 [21]. Structures of N. crassa CDH were resolved in the open-state, in which the cytochrome domain was spatially separated from the flavodehydrogenase domain. The structure of M. thermophilum CDH was resolved in the closed-state. It is suggested that the closed-state allows interdomain electron transfer from FAD to heme b, whereas the subsequent reduction of LPMO occurs in the open-state, in which the heme b cofactor is accessible. CDH is a glycosylated enzyme featuring high-mannose type N-glycosides and O-glycosylation.

Family Firsts

First CDH identified

In 1974 from the white-rot fungus Phanerochaete chrysosporium [25]

First demonstration of interaction with AA9 (LPMO)

Patent US20100159536, and later published in more detail [26]

First stereochemistry determination

The nature of the product was determined by XX... ().

First catalytic nucleophile identification

[24].

First 3-D structure

P. chrysosporium cytochrome domain was crystallized in 2000 [22] P. chrysosporium flavodehydrogenase domain was resolved in 20022 [24], first full structures were reported in 2015 in the open-state (N. crassa, [21]) and in the closed-state (M. thermophilum, [21]).

Subfamily 2 - P2O

Kinetics and Mechanism

Pyranose 2-oxidase (P2O; syn. pyranose oxidase, POX; EC 1.1.3.10; pyranose:oxygen 2-oxidoreductase) is widespread in lignin-degrading white-rot fungi and catalyzes the oxygen dependent oxidation of several monosaccharides at the C2 position to the corresponding 2-keto-aldoses and H₂O₂ [17]. The preferred substrate is D-glucose, which is oxidized to 2-keto-D-glucose. Unlike other GMC-oxidoreductases, the FAD in P2O is covalently linked via its isoalloxazine ring 8α-methyl group to the N12 atom of His167 (histidyl linkage, [27]). The supposed physiological role of P2O is to generate H₂O₂ as a substrate for lignin-degrading peroxidases. P2O is localized in the hyphal periplasmatic space [28].

During the reductive half reaction, two electrons are transferred from the substrate to the FAD, leading to the formation of FADH₂ and the 2-keto sugar. During the oxidative half reaction, the two stored electrons are transferred to molecular oxygen to form H₂O₂. The catalytic reaction is proposed to be of the ping-pong bi-bi type, since the 2-keto-sugar product is released prior to the reaction with oxygen [17]. The active site contains a conserved, catalytic His–Asn pair positioned below the FAD isoalloxazine ring, which is typical for GMC oxidoreductases. His XXX (PDB) or His YYY (PDB) is the catalytic base that facilitates deprotonation of the sugar substrate.

Catalytic Residues

The FAD cofactor in the flavodehydrogenase domain is covalently bound. The carbohydrate C2 atom is oriented towards N5 of the FAD isoalloxazine ring at a distance of approx. XY9 Å (PDB, Ref.3). Indispensible for catalysis is the general base histidine (HXXX in PDB, HYYY in PDB). A strictly conserved arginine residue (Argxxx in PDB, Arg YYY in PDB) is close to the caltalytic histidine [17].

Three-dimensional structures

Crystal structures of P2O from Trametes multicolor and Phanerochaete chrysosporium were resolved (pdb: 1TT0, [29];pdb: 4MIG; [30]) P2O is a homotetrameric enzyme with a molecular masses of approx. 68,000 per subunit. Each subunit contains the flavin-binding Rossmann-fold typical for GMC- oxidoreductases. The substrate-binding subdomain comprises six-stranded central-β sheet and three α-helices. The homotetramer features a large internal cavity, from which the four active sites are accessible. P2O is a non-glycosylated enzyme.

Family Firsts

First P2O identified

Pyranose oxidase was first isolated from the basidiomycete Polyporus obtusus in 1968 [31].

First 3-D structure

P2O from Trametes multicolor MB 49 (pdb: 1TT0; [29])

Subfamily 3 - GOx

Kinetics and Mechanism

Glucose oxidase (GOx, syn. GOD, EC 1.1.x.x., glucose:oxygen 1-oxidoreductase) catalyzes the regioselective oxidation of β-D-glucose to gluconic acid by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide. Glucose oxidases are produced by a number of fungi and insects. Proposed functions are related to the peroxide-producing abilities of the enzyme and include the preservation of honey, microbial defense as well as the support of H2O2 dependent ligninases in wood degrading fungi [32].

In the oxidative half reaction of GOx, two-electron oxidation of D-glucose at C1 results in the formation of gluconolactone and reduced cofactor (FADH₂). Reduction by glucose occurs via a hydride ion transfer to the N-5 position of the FAD. Oxygen reduction to H₂O₂ involves the uptake of two electrons (from FADH₂) and two protons. A highly conserved His (His516 in Aspergillus niger GOx) in the active site of the enzyme was identified by site directed mutagenesis as the catalytic base [33].

Catalytic Residues

Content is to be added here.

Three-dimensional structures

Content is to be added here.

Family Firsts

First GOx identified

Glucose oxidase from Aspergillus niger in 1928 [34].

First 3D structure

Glucose oxidase from Aspergillus niger (1GAL; [35]).

References

1. Levasseur A, Drula E, Lombard V, Coutinho PM, and Henrissat B. (2013). Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels. 2013;6(1):41. DOI:10.1186/1754-6834-6-41 |

2. ützl2018 pmid=29411063

4. pmid= 22249717

5. Kracher D, Scheiblbrandner S, Felice AK, Breslmayr E, Preims M, Ludwicka K, Haltrich D, Eijsink VG, and Ludwig R. (2016). Extracellular electron transfer systems fuel cellulose oxidative degradation. Science. 2016;352(6289):1098-101. DOI:10.1126/science.aaf3165 |

6. pmid= 27312718

7. Goswami P, Chinnadayyala SS, Chakraborty M, Kumar AK, and Kakoti A. (2013). An overview on alcohol oxidases and their potential applications. Appl Microbiol Biotechnol. 2013;97(10):4259-75. DOI:10.1007/s00253-013-4842-9 |
8. Iida K, Cox-Foster DL, Yang X, Ko WY, and Cavener DR. (2007). Expansion and evolution of insect GMC oxidoreductases. BMC Evol Biol. 2007;7:75. DOI:10.1186/1471-2148-7-75 |
9. Sun W, Shen YH, Yang WJ, Cao YF, Xiang ZH, and Zhang Z. (2012). Expansion of the silkworm GMC oxidoreductase genes is associated with immunity. Insect Biochem Mol Biol. 2012;42(12):935-45. DOI:10.1016/j.ibmb.2012.09.006 |

10. Mendes S, Banha C, Madeira J, Santos D, Miranda V, Manzanera M, Ventura MR, van Berkel WJH, Martins LO. Characterization of a bacterial pyranose 2-oxidase from Arthrobacter siccitolerans. 2016 J Mol Catal B Enzym 133, S34–S43