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Glycoside Hydrolase Family 26
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|Glycoside Hydrolase Family GH26|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolases of family 26 are primarily of endo-β-1,4-mannanases, although a recent exo-acting β-mannanase has been described . The family also contains enzymes that display β-1,3:1,4-glucanase  and β-1,3-xylanase activities . 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 .
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) .
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 . The identity of the catalytic nucleophile was also revealed through site-directed mutagenesis  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 . 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 .
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 . 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 , or substrates that are very slowly hydrolyzed in the wild type enzyme , 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 . 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 . The crystal structures have also revealed the mechanism of substrate recognition in subsites distal to -1 [10, 11].
- First sterochemistry determination
- Cellvibrio japonicus CjMan26A by NMR .
- First catalytic nucleophile identification
- Cellvibrio japonicus CjMan26A initially by mutagenesis and sequence conservation  and later by X-ray crystallography .
- First general acid/base residue identification
- Cellvibrio japonicus CjMan26A initially be mutagenesis, sequence conservation and kinetics mutant against activated substrate .
- First 3-D structure
- Cellvibrio japonicus CjMan26A .
- 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 |
- 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 |
- 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 |
- Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991;55(2):303-15. DOI:10.1128/mr.55.2.303-315.1991 |
- 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 |
- Ducros VM, Zechel DL, Murshudov GN, Gilbert HJ, Szabó L, Stoll D, Withers SG, and Davies GJ. (2002). Substrate distortion by a beta-mannanase: snapshots of the Michaelis and covalent-intermediate complexes suggest a B(2,5) conformation for the transition state. Angew Chem Int Ed Engl. 2002;41(15):2824-7. DOI:10.1002/1521-3773(20020802)41:15<2824::AID-ANIE2824>3.0.CO;2-G |
- Hogg D, Woo EJ, Bolam DN, McKie VA, Gilbert HJ, and Pickersgill RW. (2001). Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding. J Biol Chem. 2001;276(33):31186-92. DOI:10.1074/jbc.M010290200 |
- Money VA, Smith NL, Scaffidi A, Stick RV, Gilbert HJ, and Davies GJ. (2006). Substrate distortion by a lichenase highlights the different conformational itineraries harnessed by related glycoside hydrolases. Angew Chem Int Ed Engl. 2006;45(31):5136-40. DOI:10.1002/anie.200600802 |
- Tailford LE, Offen WA, Smith NL, Dumon C, Morland C, Gratien J, Heck MP, Stick RV, Blériot Y, Vasella A, Gilbert HJ, and Davies GJ. (2008). Structural and biochemical evidence for a boat-like transition state in beta-mannosidases. Nat Chem Biol. 2008;4(5):306-12. DOI:10.1038/nchembio.81 |
- Le Nours J, Anderson L, Stoll D, Stålbrand H, and Lo Leggio L. (2005). The structure and characterization of a modular endo-beta-1,4-mannanase from Cellulomonas fimi. Biochemistry. 2005;44(38):12700-8. DOI:10.1021/bi050779v |
- Tailford LE, Ducros VM, Flint JE, Roberts SM, Morland C, Zechel DL, Smith N, Bjørnvad ME, Borchert TV, Wilson KS, Davies GJ, and Gilbert HJ. (2009). Understanding how diverse beta-mannanases recognize heterogeneous substrates. Biochemistry. 2009;48(29):7009-18. DOI:10.1021/bi900515d |