Auxiliary Activity Family 3

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

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

Familiy members

AA3 members have been divided into three subfamilies based on activity.

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 [1]. An important in vivo function of CDH is the reduction of lytic polysaccharide monooxygenases belonging to the auxiliary activity families AA9 and AA10 [2, 3]. 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 [1, 4]. CDHs typically discriminate monosaccharides and show a low catalytic efficiency towards glucose, galactose and mannose [1, 5].

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 [6]. 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 [7]. 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 [8]. 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 [1, 5].

Based on phylogenetic, catalytic and molecular differences CDHs are classified into three classes [9]. 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 [10]. 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 [6] 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 [11].

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, [12]). 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 [13]. The crystal structure of the P. chrysosporium flavodehydrogenase domain (1.6 Å resolution, 1KDG) was reported in 2002 [14]. 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 [11]. 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 [15]

First demonstration of interaction with AA9 (LPMO)

Patent US20100159536, and later published in more detail [16]

First stereochemistry determination

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

First catalytic nucleophile identification

[14].

First 3-D structure

P. chrysosporium cytochrome domain was crystallized in 2000 [12] P. chrysosporium flavodehydrogenase domain was resolved in 20022 [14], first full structures were reported in 2015 in the open-state (N. crassa, [11]) and in the closed-state (M. thermophilum, [11]).

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 [7]. 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, [17]). 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 [18].

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 [7]. 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 (reason, Ref.).

Three-dimensional structures

Ccrystal structures of P2O from Phanerochaete chrysosporium and Trametes multicolor werte resolved (PDB, Ref.; PDB, Ref.9.) 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 [19].

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

P2O from Trametes multicolor MB 49 (pdb: 1TT0; [12])

Subfamily 3 - GO

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 [13].

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 [14].

Catalytic Residues

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Three-dimensional structures

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Family Firsts

First GOx identified

Glucose oxidase from Aspergillus niger in 1928 [20].

First 3D structure

Glucose oxidase from Aspergillus niger (1GAL; [21]).

First general acid/base residue identification

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First 3-D structure

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References

1. Zamocky M, Ludwig R, Peterbauer C, Hallberg BM, Divne C, Nicholls P, and Haltrich D. (2006). Cellobiose dehydrogenase--a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi. Curr Protein Pept Sci. 2006;7(3):255-80. DOI:10.2174/138920306777452367 |
2. Phillips CM, Beeson WT, Cate JH, and Marletta MA. (2011). Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem Biol. 2011;6(12):1399-406. DOI:10.1021/cb200351y |
3. Loose JS, Forsberg Z, Kracher D, Scheiblbrandner S, Ludwig R, Eijsink VG, and Vaaje-Kolstad G. (2016). Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase. Protein Sci. 2016;25(12):2175-2186. DOI:10.1002/pro.3043 |
4. Zhang R, Fan Z, and Kasuga T. (2011). Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization. Protein Expr Purif. 2011;75(1):63-9. DOI:10.1016/j.pep.2010.08.003 |
5. Henriksson G, Sild V, Szabó IJ, Pettersson G, and Johansson G. (1998). Substrate specificity of cellobiose dehydrogenase from Phanerochaete chrysosporium. Biochim Biophys Acta. 1998;1383(1):48-54. DOI:10.1016/s0167-4838(97)00180-5 |
6. Hallberg BM, Henriksson G, Pettersson G, Vasella A, and Divne C. (2003). Mechanism of the reductive half-reaction in cellobiose dehydrogenase. J Biol Chem. 2003;278(9):7160-6. DOI:10.1074/jbc.M210961200 |
7. Wongnate T and Chaiyen P. (2013). The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily. FEBS J. 2013;280(13):3009-27. DOI:10.1111/febs.12280 |
8. Igarashi K, Momohara I, Nishino T, and Samejima M. (2002). Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Biochem J. 2002;365(Pt 2):521-6. DOI:10.1042/BJ20011809 |
9. Zámocký M, Hallberg M, Ludwig R, Divne C, and Haltrich D. (2004). Ancestral gene fusion in cellobiose dehydrogenases reflects a specific evolution of GMC oxidoreductases in fungi. Gene. 2004;338(1):1-14. DOI:10.1016/j.gene.2004.04.025 |
11. Tan TC, Kracher D, Gandini R, Sygmund C, Kittl R, Haltrich D, Hällberg BM, Ludwig R, and Divne C. (2015). Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation. Nat Commun. 2015;6:7542. DOI:10.1038/ncomms8542 |
12. Hallberg BM, Bergfors T, Bäckbro K, Pettersson G, Henriksson G, and Divne C. (2000). A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase. Structure. 2000;8(1):79-88. DOI:10.1016/s0969-2126(00)00082-4 |
13. Cox MC, Rogers MS, Cheesman M, Jones GD, Thomson AJ, Wilson MT, and Moore GR. (1992). Spectroscopic identification of the haem ligands of cellobiose oxidase. FEBS Lett. 1992;307(2):233-6. DOI:10.1016/0014-5793(92)80774-b |
14. Hallberg BM, Henriksson G, Pettersson G, Vasella A, and Divne C. (2003). Mechanism of the reductive half-reaction in cellobiose dehydrogenase. J Biol Chem. 2003;278(9):7160-6. DOI:10.1074/jbc.M210961200 |

15. Westermark U & Eriksson K-E Cellobiose:Quinone Oxidoreductase, a New Wood-degrading Enzyme from White-rot Fungi. Acta Chem. Scand. 1974 28b, 209–214.

16. Langston JA, Shaghasi T, Abbate E, Xu F, Vlasenko E, and Sweeney MD. (2011). Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl Environ Microbiol. 2011;77(19):7007-15. DOI:10.1128/AEM.05815-11 |
17. Sucharitakul J, Prongjit M, Haltrich D, and Chaiyen P. (2008). Detection of a C4a-hydroperoxyflavin intermediate in the reaction of a flavoprotein oxidase. Biochemistry. 2008;47(33):8485-90. DOI:10.1021/bi801039d |
18. Giffhorn F (2000). Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry. Appl Microbiol Biotechnol. 2000;54(6):727-40. DOI:10.1007/s002530000446 |
19. Ruelius HW, Kerwin RM, and Janssen FW. (1968). Carbohydrate oxidase, a novel enzyme from Polyporus obtusus. I. Isolation and purification. Biochim Biophys Acta. 1968;167(3):493-500. DOI:10.1016/0005-2744(68)90039-9 |

20. Mueller D Oxidation von Glukose mit Extrakten aus Aspegillus niger. Biochem Z 199, 136–170

21. Hecht HJ, Kalisz HM, Hendle J, Schmid RD, and Schomburg D. (1993). Crystal structure of glucose oxidase from Aspergillus niger refined at 2.3 A resolution. J Mol Biol. 1993;229(1):153-72. DOI:10.1006/jmbi.1993.1015 |