Auxiliary Activity Family 3

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• Author: ^^^Roland Ludwig^^^
• Responsible Curator: ^^^Roland Ludwig^^^

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Auxiliary Activity Family AA3
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://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) is a flavohaemoprotein (or flavocytochrome) secreted by lignocellulose degrading, phytopathogenic and saprotrophic fungi belonging to the phyla of Basidiomycota and Ascomycota (Zamocky et al. 2006). An important in vivo function of CDH is the reduction of lytic polysaccharide monooxygenases belonging to the auxiliary activity families AA9 and AA10 (Marletta ACS Chem Biol., Eiksink protein Science). 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 (Zamocky et al. 2006; N. crassa CDH kalifornische Japaner). CDHs typically discriminate monosaccharides and show a low catalytic efficiency towards glucose, galactose and mannose (Henriksson et al. 1998).

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 (Hallberg et al. 2003). 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 (Chaiyen et al. XXXX). 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 [1,2, Igarashi et al. 2002]. 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 (Ref. Zamocky et al. Henriksson et al.).

Based on phylogenetic, catalytic and molecular differences CDHs are classified into three classes (Zamocky et al. 2004 Gene). 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 (Classification of asc CDH et el.). No member From the phylogenetic group of Class-III CDHs has been characterized yet. and -III CDHs).

Catalytic Residues

The FAD cofactor in the flavodehydrogenase domain is noncovalently bound. The substrates anomeric carbon atom is oriented towrds N5 of the FAD isoalloxazine ring at a distance of approx. 2.9 Å (Hallberg et al. 2003) and approx. xy Å from 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 electgron transfer is R7XX (Ref. Nature Commun).

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 (1D7C, XXX, Hallberg et al., 2000). 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 (Cox et al., 1992). The crystal structure of the P. chrysosporium flavodehydrogenase domain (1.6 Å resolution, 1KDG) was reported in 2002 (Hallberg et al.). 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. 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.

Family Firsts

First CDH identified

In 1974 from the white-rot fungus Trametes versicolor Phanerochaete chrysosporium [3]

First demonstration of interaction with AA9 (LPMO)

https://www.google.com/patents/US20100159536, and later published in more detail [4].;First stereochemistry determination: The nature of the product was determined by XX... ().

First catalytic nucleophile identification

Hallberg 2002.

First general acid/base residue identification

CHallberg 2002.

First 3-D structure

Single domains: P. chrysosporium cytochrome domain (Halberg et al. 2000) P. chrysosporium flavodehydrogenase domain 2002 (Hallberg et al. 2002), first full structure open-state (N. crassa, Nat. Commun. 2015), closed-state (M. thermophilum, Nat. Commun. 2015).

Subfamily 2 - P2O

Kinetics and Mechanism

P2O (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 yield the corresponding 2-keto-aldoses and H2O2 [8]. The preferred substrate is D-glucose, which is oxidized to 2-keto-D-glucose. The FAD cofactor in P2O is covalently linked through a histidyl linkage. The supposed physiological role of P2O is to generate H2O2, which is a substrate for lignin-degrading peroxidases. P2Os are localized in the hyphal periplasmatic space [9].

During the reductive half reaction, 2 electrons are transferred from the substrate to the FAD, leading to the formation of FADH2 and a 2-keto sugar. During the oxidative half reaction, reduction equivalents are transferred to molecular oxygen to yield H2O2. The catalytic reaction can be classified as a ping-pong bi-bi type, since the 2-keto-sugar product is released prior to the reaction with oxygen. P2O is the only known flavin-dependent oxidase which generates a C4a-hydroperoxy-flavin intermediate during the oxidative half-reaction [10]. Unlike other GMC enzymes, the FAD in P2O is covalently linked via its 8α-methyl group to the N12 atom of His167. The active site contains a conserved, catalytic His–Asn pair positioned below the FAD isoalloxazine ring, which is typical for GMC oxidoreductases. His is the catalytic base that facilitates deprotonation of the sugar substrate.

Catalytic Residues

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Three-dimensional structures

Several crystal structure of P2O from P. chrysosporium and Trametes multicolor with various ligands are available in the pdb database. P2Os are homotetrameric enzymes with native molecular masses of approx. 270 kDa containing 4 flavin-binding Rossmann domains of class α/β typical for GMC oxidoreductases. The substrate-binding subdomain has a six-stranded central β sheet and three α helices. The homotetramer conceals a large internal cavity, from which the four active sites are accessible. Four substrate channels lead from the protein surface to the active sites.

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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First P2O identified

Pyranose oxidase was first isolated from the basidiomycete Polyporus obtusus in 1968 [11].

First 3-D structure

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

Subfamily 3 - GO

Kinetics and Mechanism

Glucose oxidase 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 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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First GOx identified

Glucose oxidase from Aspergillus niger in 1928 (Müller D (1928) Oxidation von Glukose mit Extrakten aus Aspegillus niger. Biochem Z 199:136–170)

First 3-D structure

Glucose oxidase from Aspergillus niger ([15], pdb code 1GAL)

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Authors may get an idea of what to put in each field from Curator Approved Glycoside Hydrolase Families. (TIP: Right click with your mouse and open this link in a new browser window...)

In the meantime, please see these references for an essential introduction to the CAZy classification system: [1, 2].

References

1. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.

2. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |