Glycoside Hydrolase Family 26

Glycoside Hydrolase Family GH26
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH26.html

Substrate specificities

This family consists primarily of endo-beta1,4-mannanases, although a recent exo-acting beta- mannanase has been described [1]. The family also contains enzymes that display beta-1,3:1,4-glucanase [2] and beta-1,3 xylanase activities [3].

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) [4].

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 [4]. The identity of the catalytic nucleophile was also revealed through site-directed mutagenesis [4] and its function was visualized by X-ray crystallography in which it was bound to 2-deoxy-2-fluoromannose in the acid-base mutant [5]. 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 [5].

Three-dimensional structures

Three-dimensional structures are available for a large number of Family GH26 enzymes, the first solved being that of the Cellvibrio japonicus (previously called various names in the genus Pseudomonas) mannanase CjMan26A [6]. As members of Clan GHA they have a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of beta-strands 4 (acid/base) and 7 (nucleophile). The crystal structure of two C. japonicus mannanases in complex with activated substrates in the acid base mutant [5], or substrates that are very slowly hydrolyzed in the wild type enzyme [1], show that catalysis by this class of enzyme proceeds via a Boat2,5 (B2,5) transition state, while the GH26 beta-1,3:1,4-glucanase transition state adopts a half-chair 4H3 conformation [7]. The chemical rationale for the different transition states adopted by beta mannanases and glucanases is discussed by Davies and colleagues in these publications and elsewhere [8]. The crystal structures have also revealed the mechanism of substrate recognition in subsites distal of -1 [9, 10].

Family Firsts

First sterochemistry determination: Cellvibrio japonicus CjMan26A by NMR [4].
First catalytic nucleophile identification: Cellvibrio japonicus CjMan26A initially by mutagenesis and sequence conservation [4] and later by X-ray crystallography [5].
First general acid/base residue identification: Cellvibrio japonicus CjMan26A initially be mutagenesis, sequence conservation and kinetics mutant against activated substrate [4].
First 3-D structure: Cellvibrio japonicus CjMan26A [6].

References

All Medline abstracts: PubMed