Glycoside Hydrolase Family 18

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Author: ^^^Gideon Davies^^^
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 cite>Tews1996. More recetly, a similar conformation has been observed for the Michaelis complex of another neighboring group enzymem 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 feauture of GH18 are the latge numbers of sequences that encode catalytically-inactive proteins that function as enzyme inhibitors or lectins. SOMEONE ELSE WRITE THIS?

Daan Chilectin paper [12] Hennig Concanavaline B paper [13] Narbonin [14]

Review of non-catalytic GH18 as enzyme inhibitors [15].

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 [16].
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, althoigh it is has no enzymatic activity [14].

References

All Medline abstracts: PubMed