Auxiliary Activity Family 14

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• Author: ^^^Marie Couturier^^^ and ^^^Jean-Guy Berrin^^^
• Responsible Curator: ^^^Jean-Guy Berrin^^^

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Auxiliary Activity Family AA14
Clan	Structurally related to AA9
Mechanism	lytic oxidase
Active site residues	mononuclear copper ion
CAZy DB link
https://www.cazy.org/AA14.html

Substrate specificities

The gene encoding the first AA14 family member was identified by analysing transcriptomic and proteomic data from the white-rot basidiomycete Pycnoporus coccineus [1]. This gene was highly upregulated when the fungus was grown on pine or poplar. The corresponding protein (GenBank ID KY769370) was secreted only during growth on pine and poplar, suggesting a role in wood decay. AA14 modules never occur with CBMs, carbohydrate-binding modules which explains why the family could not be discovered by the module-walking approach, as were AA11 and AA13.

The only two AA14 characterized so far were tested for copper dependant oxidase activity on a range of polysaccharides. No activity could be detected on any substrate tested, including cellulose and xylans. However, addition of either of the AA14 enzymes to a Trichoderma reesei cocktail composed of mainly cellulases and xylanases led to a boost of glucose release from poplar and pine . This improvement in glucose release was dose dependent, yielding up to ~100% increase on pretreated softwood. AA14 enzymes also showed synergystic action on wood with AA9 LPMOs. Finally, activity was detected on xylan adsorbed onto cellulose chains, using solid state 13C CP/MAS NMR and mass spectrometry. The observed products were C1 oxidized species with an aldonic acid at the reducing end.

Kinetics and Mechanism

As all LPMOs, AA14s are copper dependent mono-oxygenases and accordingly, mass spectrometry analyses revealed that PcAA14A and PcAA14B contained ∼1 copper atom per protein molecule. However, as for the other LPMOs, the chemical mechanism by which the enzymes perform the reaction is still a matter of debate [2],[3]. Although the natural electron donor for AA14s is unknown, PcAA14A and PcAA14B were both able to produce hydrogen peroxide in the presence of ascorbate, cysteine or gallate as electron donors. They were also active on micronized wood without addition of electron donor, suggesting that wood components such as lignin may act as electron donors (Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer [4]. N-terminal histidine in AA14s is methylated as seen for all other fungal LPMOs, but the effect on the reaction performed by the enzyme is not established yet.

Catalytic and other important Residues

AA14s exhibit the canonical histidine brace, exposed at the surface, that coordinates the copper ion. In PcAA14B this histidine brace is constituted by His1, His99 and Tyr176. Interestingly, PcAA14B possesses an equally conserved tyrosine residue, Tyr240, at the edge of the substrate-binding surface, albeit located on a different loop region, which could potentially make substrate interactions. This tyrosine residue is also conserved in AA9 [5]

Three-dimensional structures

The structure of PcAA14B was solved by multiple-wavelength anomalous dispersion data recorded at the gadolinium edge, and refined at 3.0 Å resolution [6]. The core of PcAA14B structure folds into a largely antiparallel immunoglobulin-like β-sandwich, a fold globally similar to those seen in LPMOs from other families [7]. However, in contrast to the flat substrate-binding surfaces observed in AA9 LPMOs, the surface of PcAA14B has a rippled shape with a clamp formed by two prominent surface loops located at the N-terminal half of the enzyme. It is interesting to note that these loops are equivalent to the L2 and L3 loop regions in AA9 LPMOs which have been shown to be involved in LPMO-substrate interactions [7].

Family Firsts

First family member identified

AA14 from Pycnoporus coccineus [6].

First demonstration of oxidative cleavage

PcAA114A and PcAA114AB were shown to oxidatively cleave xylan chains bound to cellulose [6].

First 3-D structure

PcAA14B from P. coccineus 5NO7 [6]

References

1. Couturier M, Navarro D, Chevret D, Henrissat B, Piumi F, Ruiz-Dueñas FJ, Martinez AT, Grigoriev IV, Riley R, Lipzen A, Berrin JG, Master ER, and Rosso MN. (2015). Enhanced degradation of softwood versus hardwood by the white-rot fungus Pycnoporus coccineus. Biotechnol Biofuels. 2015;8:216. DOI:10.1186/s13068-015-0407-8 |
2. Hedegård ED and Ryde U. (2018). Molecular mechanism of lytic polysaccharide monooxygenases. Chem Sci. 2018;9(15):3866-3880. DOI:10.1039/c8sc00426a |
3. Bertini L, Breglia R, Lambrughi M, Fantucci P, De Gioia L, Borsari M, Sola M, Bortolotti CA, and Bruschi M. (2018). Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations. Inorg Chem. 2018;57(1):86-97. DOI:10.1021/acs.inorgchem.7b02005 |
4. Westereng B, Cannella D, Wittrup Agger J, Jørgensen H, Larsen Andersen M, Eijsink VG, and Felby C. (2015). Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer. Sci Rep. 2015;5:18561. DOI:10.1038/srep18561 |
5. Frandsen KE, Simmons TJ, Dupree P, Poulsen JC, Hemsworth GR, Ciano L, Johnston EM, Tovborg M, Johansen KS, von Freiesleben P, Marmuse L, Fort S, Cottaz S, Driguez H, Henrissat B, Lenfant N, Tuna F, Baldansuren A, Davies GJ, Lo Leggio L, and Walton PH. (2016). The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat Chem Biol. 2016;12(4):298-303. DOI:10.1038/nchembio.2029 |
6. Couturier M, Ladevèze S, Sulzenbacher G, Ciano L, Fanuel M, Moreau C, Villares A, Cathala B, Chaspoul F, Frandsen KE, Labourel A, Herpoël-Gimbert I, Grisel S, Haon M, Lenfant N, Rogniaux H, Ropartz D, Davies GJ, Rosso MN, Walton PH, Henrissat B, and Berrin JG. (2018). Lytic xylan oxidases from wood-decay fungi unlock biomass degradation. Nat Chem Biol. 2018;14(3):306-310. DOI:10.1038/nchembio.2558 |
7. Vaaje-Kolstad G, Forsberg Z, Loose JS, Bissaro B, and Eijsink VG. (2017). Structural diversity of lytic polysaccharide monooxygenases. Curr Opin Struct Biol. 2017;44:67-76. DOI:10.1016/j.sbi.2016.12.012 |