Glycoside Hydrolase Family 26

Substrate specificities

This family consists primarily of endo-beta1,4-mannanases, although a recent exo-acting beta- mannanase has been described. The family also contains enzymes that display beta-1,3:1,4-glucanase [1] and beta-1,3 xylanase activities.

Kinetics and Mechanism

Family GH26 enzymes are “retainers”, as shown by NMR and follow a classical Koshland double-displacement mechanism. Pre-steady state kinetics using activated substrates revealed the two phases of the reaction; the rapid initial glycosylation step (only with good leaving groups) followed by the slower deglycosylation. It should be noted that the use of substrates with a good leaving group result in a very low apparent KM, particularly with the acid-base mutant. This does not reflect tight affinity but simply that the glycosylation step (k2) is much quicker than the deglycosylation step (k3) {Bolam, 1996 #7}.

Catalytic Residues

The catalytic residues were first identified in the endo-beta1,4-mannanase CjMan26A. The catalytic acid-base is the glutamate Glu320, which is separated in sequence by ~100 residues from the catalytic nucleophile, Glu212. The catalytic nucleophile was identified by site-directed mutagenesis in harness with the kinetics of 2,4-dintrophenyl-beta-mannobioside hydrolysis which, although very slow was associated with a dramatic decrease in KM [2, 3, 4]. The identity of the catalytic nucleophile was also revealed through site-directed mutagenesis {Bolam, 1996 #7} and its function was visualized by X-ray crystallography in which it was bound to 2-deoxy-2-fluoromannose in the acid-base mutant {Ducros, 2002 #45}. In Clan GHA, of which GH26 is a member, the residue immediately preceding the acid base in sequence is an asparagine that makes pivotal interactions with the 2-hydroxyl of the substrate. In GH26 the equivalent amino acid is a histidine, His211 in CjMan26A, although its function is conserved; it also makes important interactions with the 2-hydroxyl of the substrate {Ducros, 2002 #45}.

Family Firsts

First sterochemistry determination

Cite some reference here, with a short explanation [1].

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

References

1. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |
5. Araki T, Hashikawa S, and Morishita T. (2000). Cloning, sequencing, and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214. Appl Environ Microbiol. 2000;66(4):1741-3. DOI:10.1128/AEM.66.4.1741-1743.2000 |