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
https://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].

Substrate Specificities

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Authors may get an idea of what to put in each field from Curator Approved Auxiliary Activity Families and 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: [13, 14].

Kinetics and Mechanism

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Catalytic Residues

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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, 15] 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 REF CMB32 PAGE) 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 [15]. Other characterized AA5_2 enzymes from Fusarium species contain CBM32 [4, 16, 17, 18], 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 [19].

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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References

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7. Mathieu, Y., Offen, W. A., Forget, S. M., Ciano, L., Viborg, A. H., Blagova, E., Henrissat, B., Walton, P.H, Davies, G.J, and Brumer, H. (2020). Discovery of a fungal copper radical oxidase with high catalytic efficiency toward 5-hydroxymethylfurfural and benzyl alcohols for bioprocessing. ACS Catalysis, 10(5), 3042-3058. https://pubs.acs.org/doi/abs/10.1021/acscatal.9b04727

10. Ögel, Z. B.; Brayford, D.; McPherson, M. J., (1994). Cellulose-triggered sporulation in the galactose oxidase-producing fungus Cladobotryum (Dactylium) dendroides NRRL 2903 and its re-identification as a species of Fusarium. Mycol. Res., 98 (4), 474-480. https://doi.org/10.1016/j.pep.2014.12.010

13. 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.

14. 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 |