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Glycoside hydrolases of family 26 are primarily of endo-β-1,4-mannanases, although a recent exo-acting β-mannanase has been described [1]. The family also contains enzymes that display β-1,3:1,4-glucanase [2] and β-1,3-xylanase activities [3]. As a historical note, GH26 was one of the first glycoside hydrolase families classified by sequence analysis, and was previously known as "Cellulase Family I (eye)" prior to detailed enzymological characterization [4].
Kinetics and Mechanism
Family GH26 enzymes utlize a retaining mechanism, 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) [5].
Catalytic Residues
The catalytic residues were first identified in the endo-β-1,4-mannanase CjMan26A. The general acid/base residue 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-β-mannobioside hydrolysis which, although very slow was associated with a dramatic decrease in KM [5]. The identity of the catalytic nucleophile was also revealed through site-directed mutagenesis [5] and its function was visualized by X-ray crystallography in the acid-base mutant of which the glycosyl enzyme intermediate bound to 2-deoxy-2-fluoromannose was formed [6]. In Clan GHA, of which GH26 is a member, the residue immediately preceding the general acid/base residue 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 [6].
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 [7]. 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 β-strands 4 (acid/base) and 7 (nucleophile). The crystal structure of two C. japonicus mannanases in complex with activated substrates with the acid base mutant [6], 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, whereas the GH26 β-1,3:1,4-glucanase transition state adopts a half-chair 4H3 conformation [8]. The chemical rationale for the different transition states adopted by β-mannanases and glucanases is discussed by Davies and colleagues in these publications and elsewhere [9]. The crystal structures have also revealed the mechanism of substrate recognition in subsites distal to -1 [10, 11].
