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Auxiliary Activity Family 14
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|Auxiliary Activity Family AA14|
|Clan||Structurally related to AA9|
|Active site residues||mononuclear copper ion|
|CAZy DB link|
Discovery of AA14 LPMOs
The gene encoding the first AA14 family member was identified by analysing transcriptomic and proteomic data from the white-rot basidiomycete Pycnoporus coccineus . 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-dependent 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 mainly composed of 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. Additionally, RPE analysis revealed spin-Hamiltonian parameters similar to those obtained for AA9 LPMOs, confirming the presence of the copper(II) ion within the histidine brace coordination environment. However, as for the other LPMOs, the chemical mechanism by which the enzymes perform the reaction is still a matter of debate [2, 3, 4]. 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 . N-terminal histidine in AA14s is methylated in vivo 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 LPMO histidine brace coordinating the copper ion, which is exposed at the surface. 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 LPMOs 
The structure of PcAA14B was solved by multiple-wavelength anomalous dispersion data recorded at the gadolinium edge, and refined at 3.0 Å resolution . 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 . 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 .
- First family member identified
- AA14 from Pycnoporus coccineus .
- First demonstration of oxidative cleavage
- PcAA114A and PcAA114AB were shown to oxidatively cleave xylan chains bound to cellulose .
- First 3-D structure
- PcAA14B from P. coccineus 5NO7 
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