Auxiliary Activity Family 9

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• Author: ^^^Paul Harris^^^
• Responsible Curator: ^^^Paul Harris^^^

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Auxillary Activity Family AA9
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
https://www.cazy.org/AA9.html

Substrate specificities

This family was originally placed among the glycoside hydrolases in family 61 based largely on a report of very weak endoglucanase activity of “Cel61A” from Trichoderma reesei using substrates such as carboxymethyl cellulose and ß-glucan. Subsequently, other investigations seemed to confirm this result with other AA9 proteins. It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active AA10 (formerly CBM33) proteins, and the ability of some AA10 proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose.

Current thinking posits that AA9 and AA10 proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of AA10 versus stacking with planar aromatic residues for AA9. Soluble polysaccharides have not been shown to be a substrate for AA9 proteins.

Kinetics and Mechanism

It was known for many years from the patent literature (e.g. US 20060005279, US20070077630, WO2005074647) and conference proceedings that some AA9 proteins could dramatically enhance the activity of canonical cellulase mixtures in the hydrolysis of lignocellulose, however this information was not published in a peer-reviewed journal until 2010. An explanation for this enhancement remained elusive for many years, but a first clue was the demonstration in the 2010 publication that metal ion is required for the enhancement and that the enhancement is not evident with relatively pure cellulose (Avicel) but only with pretreated lignocellulose such as acid-pretreated corn stover. In hindsight it became clear that the complex lignocellulosic substrate was providing an essential cofactor(s) that the Avicel did not. Subsequent studies, again first published in the patent literature (US 20100159536) and subsequently in journals, showed that a combination of AA9 and cellobiose dehydrogenase could cleave pure cellulose. Additional work showed that a large number of small redox-active molecules such as gallate, ascorbate and catechol could replace cellobiose dehydrogenase in this regard, and both soluble and insoluble lignin and lignin derivatives might also act in this capacity. However, the “natural” redox-active cofactors for AA9 proteins are currently unknown and may well vary depending on availability and the specific AA9 protein. Cellobiose dehydrogenase is often mentioned as a potential natural cofactor and indeed is often co-secreted with AA9 proteins. However there are many species of filamentous fungi (e.g. Paxillus involutus, Talaromyces stipitatus, Talaromyces thermophilus, and several Trichoderma (Hypocrea) species) that do not produce cellobiose dehydrogenase (based on lack of an obvious gene encoding it) and yet have multiple AA9-encoding genes. Nonetheless, for those fungi that do secrete cellobiose dehydrogenase, one of its main functions may be to synergize with AA9 proteins, presumably by acting as an electron donor.

A key publication in 2010 showed that AA10, a structural relative of AA9, could oxidatively degrade crystalline chitin in the presence of metal ion and the reductant ascorbate, producing a mixture of oxidized and unoxidized oligosaccharides. Shortly thereafter, similar data were published on AA9 showing that these proteins are copper-dependent monooxygenases which are capable of inserting oxygen at the C-1 or C-4 (and perhaps C-6) position of the glycosidic bond in cellulose. The so-called type 1 LPMOs produce predominantly oxidation at C-1 (reducing end), probably initially producing an aldonolactone that spontaneously hydrolyzes to an aldonic acid. Type 2 LPMOs generate predominantly C-4 non-reducing end oxidized products (4-ketoaldolase), and type 3 LPMOs appear to produce oxidized products at both the reducing and nonreducing ends. Results consistent with C-6 oxidation to a 6-ketal sugar (geminal diol) have also been reported. Positioning of the substrate on the catalytic surface very likely determines the site of oxidation. The detailed interactions between AA9, bound metal ion, redox-active cofactor and substrate remain an area of active investigation, and additional surprises are likely. At present, the ability of AA9 proteins to oxidize cellulose is thought to arise from generation of oxidizing copper-oxygen species (superoxides or peroxides) at the active site by a mechanism that remains unclear. Presumably Cu(I) at the active site activates a molecular oxygen and the activated oxygen species becomes incorporated into the cellulose chain. A reductant is then necessary to regenerate Cu(I).

Catalytic Residues

AA9 proteins are quite diverse in sequence with identities between some sequences below 30% within the main catalytic domain. However, the catalytic copper ion is coordinated by a highly conserved “histidine brace” consisting of the imidazole and backbone amino group of the N-terminal histidine and the imidazole of an additional histidine. Closely associated with the catalytic copper in most AA9 proteins is the phenolic oxygen of a highly conserved tyrosine that could play a redox role in catalysis since mutation to phenylalanine greatly reduces but does not entirely eliminate activity in “GH61E” from Thielavia terrestris. About 3% of all sequenced AA9 proteins and the majority of AA10 proteins naturally contain phenylalanine at this position rather than tyrosine. Additional contributions to catalysis might come from long distance internal electron transfer through a conserved ionic network and conserved aromatic residues, although this has not yet been directly demonstrated.

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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References

1. 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 |

2. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). DOI: 10.1042/BJ20080382