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 [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) [1].

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, 5, 6]. 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}.

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 {Hogg, 2001 #10}. 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 {Ducros, 2002 #45}, or substrates that are very slowly hydrolyzed in the wild type enzyme {Cartmell, 2008 #53}, 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 configuration {Money, 2006 #23}. 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 {Tailford, 2008 #57}. The crystal structures have also revealed the mechanism of substrate recognition in subsites distal of -1 {Le Nours, 2005 #63} {Tailford, 2009 #64}.

Family Firsts

First sterochemistry determination

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Cellvibrio japonicus CjMan26A by NMR [4, 5, 6]

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

References

1. Bolam DN, Hughes N, Virden R, Lakey JH, Hazlewood GP, Henrissat B, Braithwaite KL, and Gilbert HJ. (1996). Mannanase A from Pseudomonas fluorescens ssp. cellulosa is a retaining glycosyl hydrolase in which E212 and E320 are the putative catalytic residues. Biochemistry. 1996;35(50):16195-204. DOI:10.1021/bi961866d |
2. 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 |
3. 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 |