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, 3] and beta-1,3 xylanase activities [4].

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) [5].

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 [6, 7, 8]. 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 [6, 7, 8]

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

References

1. Cartmell A, Topakas E, Ducros VM, Suits MD, Davies GJ, and Gilbert HJ. (2008). The Cellvibrio japonicus mannanase CjMan26C displays a unique exo-mode of action that is conferred by subtle changes to the distal region of the active site. J Biol Chem. 2008;283(49):34403-13. DOI:10.1074/jbc.M804053200 |
4. 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 |
5. 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 |
9. Taylor EJ, Goyal A, Guerreiro CI, Prates JA, Money VA, Ferry N, Morland C, Planas A, Macdonald JA, Stick RV, Gilbert HJ, Fontes CM, and Davies GJ. (2005). How family 26 glycoside hydrolases orchestrate catalysis on different polysaccharides: structure and activity of a Clostridium thermocellum lichenase, CtLic26A. J Biol Chem. 2005;280(38):32761-7. DOI:10.1074/jbc.M506580200 |