Glycoside Hydrolase Family 18

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Authors: ^^^Gideon Davies^^^, ^^^Nathalie Juge^^^
Responsible Curator: ^^^Gideon Davies^^^

Glycoside Hydrolase Family GH18
Clan	GH-K
Mechanism	retaining
Active site residues	known (acid/neighbouring group)
CAZy DB link
http://www.cazy.org/fam/GH18.html

Substrate specificities

GH18 is unusual in having both catalytically active chitinase (EC 3.2.1.14) and endo-β-N-acetylglucosaminidases (EC 3.2.1.96) but there are also sub-families of non-hydrolytic proteins that function as carbohydrate binding modules / "lectins" or as xylanase inhibitors.

Kinetics and Mechanism

GH18 enzymes belong to a growing group of enzymes (now including GH families 18, 20,25, 56, 84, and 85) that perform a double-displacement reaction but instead of the more common enzyme-derived nucleophile they utlize the N-acetamido carbonyl oxygen in what is termed "neighbouring group participation" / "substrate participation" or "anchimeric assistance". Figures showing such a mechanism date back to Koshland's 1953 review [1], indeed they frequent the chemical literature of participating groups long before that, but it is primarily through the work on GH18 [2] and soon after GH20 [3, 4] that such a mechanism became well established. In such a mechanism, which occurs with (net) retention of anomeric configuration, the enzyme provides a catalytic acid function to protonate the leaving group to facilitate its departure with the substrate carbonyl oxygen playing the role of nucleophile to generate a bicyclic "oxazoline" intermediate (which subsequently breaks down following the microscopic reverse via hydrolysis or occasionally transglycosylation). Such a mechanism has a number of facets, one of which is its potential inhibition using thiazolines [5].

Catalytic Residues

The catalytically active GH18 enzymes use a double displacement reaction mechanism with "neighbouring group participation". Hence there is a catalytic acid residue (glutamate in family GH18, but often also Asp in other families using this mechanism) and in all families apart from GH85 (where this residue is an amide), a second carboxylate (here Asp) acts to deprotonate the N-acetamido nitrogen during oxazoline formation/breakdown. In family GH18 the two catalytic carboxylates are found in an D-X-G motif whereas in some families the carboxylates may be adjacent such as the DD motif in family GH84 (for example see [6]). The physical separation of the two catalytic residues (with the second not in a position to act as a nucleophile itself) has led to confusion in some literature that GH18 and other enzymes (notably GH25) may be inverting enzymes; this is certainly not the case for GH18 and is unlikely to be the case for GH25.

Three-dimensional structures

Although these enzymes are frequently multi-modular, the catalytic domains are α / β barrels [7, 8].

Work on the conformational itinerary of catalysis which is extremely similar to other retaining enzymes active on gluco-configured substrates, was provided by the van Aalten group [9] in 2001 through the trapping of a distorted Michaelis complex in ^1,4B conformation and thus extremely similar to the ¹S₃ skew boats oberserved in GH5 [10] for example or the ⁴E conformation originally seen for a "neighboring group" enzyme in GH20 [3]. More recently, a similar conformation has been observed for the Michaelis complex of another neighboring group enzyme, the GH84 O-GlcNAcase [6]. Fungal GH18 enzymes are considered as possible therapeutic targets and a number of programmes are probing this area (for example [11]).

One unusual of feature of plant members of the GH18 family is the large number of sequences that encode catalytically-inactive proteins that function as enzyme inhibitors or lectins. Phylogenetic analysis of the plant GH18 family reveals clear distinction between hevamine-type chitinases, putative chitinases and narbonins [12]. Out of the major subfamilies, only the one that contains hevamine actually contains enzymes of demonstrated activity [13]. The subfamily of GH18 coding for xylanase inhibitor proteins (XIP) emerged from the hevamine cluster along with concanavalin B. All have nonconservative substitutions of one of the acidic amino acid residues in the catalytic region. In the structure of concanavalin B the catalytic Glu residue is replaced by Gln [14], which mostly account for the lack of chitinase activity reported for this protein. The XIP-type inhibitors all have the third aspartic acid DxxDxDxE mutated into an aromatic residue whereas the catalytic glutamate residue is only conserved in the prototype of cereal xylanase inhibitors, XIP-I (isolated from Triticum aetivum) [15]. In XIP-I and narbonin, the glutamic acid residues are present in an equivalent position to the catalytic residue in hevamine, but their side chain is fully engaged in salt bridges with neighbouring arginine residues [16], preventing chitinase activity despite the presence of the catalytic residue. Furthermore in both XIP-I and narbonin, the position equivalent to residue Asp in hevamine, which has been proposed to stabilize the positively charged oxazoline reaction intermediate ([13]), is occupied by a bulky residue[16]. The mutation of this Asp residue in alanine in hevamine led to a mutant with approx. 2% residual activity [17]. The most striking disruption of the cleft in XIP-I and narbonin is caused by the mutation of subsite -1 Gly which participates in the hydrogen-bonding network with the ligand [18, 19], resulting in complete obstruction of subsite -1and preventing access to the catalytic residue [20]. Xylanase inhibitors appeared after the emergence of the various subfamilies of chitinases from their common ancestor. In this respect, the xylanase inhibitors are a relatively new invention, and so far no protein has been reported to display both xylanase inhibition and chitinase activities. GH18 XIP-type inhibitors can inhibit xylanases from GH10 and GH11 families [15]. The inhibition specificity of the GH18 xylanase inhibitors can be explained on the basis of the solved 3-D structure of XIP-I in complex with a GH10 xylanase from A. nidulans and a GH11 xylanase from P. funiculosum [21].

Family Firsts

First sterochemistry determination: Sometimes incorrectly reported as inverting, this family performs catalysis with retention of anomeric configuration as first shown on the Bacillus ciculans enzyme [22].
First catalytic nucleophile identification: This family is one of many that uses neighbouring group participation for catalysis with the N-acetyl carbonyl group acting as the nucleophile; first proposed (I believe) for this family in [2].
First general acid/base residue identification: On the basis of 3-D structure [7].
First 3-D structure: The first two 3-D structures for catalytically active GH18 members were the Serratia marcescens chitinase A and the plant defence protein hevamine published "back-to-back" in Structure in 1994 [7, 8]. In retrospect, however, the non-catalytic "Narbonin" structure was arguably the first GH18 3-D structure, although it is has no enzymatic activity [23].

References

All Medline abstracts: PubMed