Glycoside Hydrolase Family 17
Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail, or using this form.
|Glycoside Hydrolase Family GH17|
|Active site residues||known|
|CAZy DB link|
The family GH17 glycoside hydrolases are clan GH-A enzymes from bacteria, fungi and plants, and include two major groups of enzymes with related but distinct substrate specificities, namely 1,3-β-D-glucan endohydrolases (EC 22.214.171.124) and 1,3;1,4-β-D-glucan endohydrolases (EC 126.96.36.199). A 1,3-β-D-glucan exohydrolase (EC 188.8.131.52) is also classified in this family. The family GH17 enzymes have quite distinct amino acid sequences and 3D structures compared with the 1,3-β-D-glucan endohydrolases and 1,3;1,4-β-D-glucan endohydrolases that have similar substrate specificities but are classified in families GH16, GH55, GH64 and GH81.
The family GH17 1,3-β-D-glucan endohydrolases hydrolyse internal 1,3-β-D-glucosidic linkages in polysaccharides, but usually require a region of contiguous unbranched, un-substituted 1,3-β-D-glucosyl residues for activity. The enzymes release 1,3-β-D-oligoglucosides of DP 2-5 as their major products. Because the 1,3-β-D-glucan endohydrolases require a region of contiguous unbranched, unsubstituted 1,3-β-D-glucosyl residues for activity, they are unable to hydrolyse the single 1,3-β-D-glucosidic linkages in 1,3;1,4-β-D-glucans from the Poaceae, but they will hydrolyse 1,3-β-D-glucosidic linkages in fungal 1,3;1,6-β-D-glucans, provided an appropriate region of contiguous un-substituted 1,3-β-D-glucosyl residues is available. The family GH17 1,3;1,4-β-D-glucan endohydrolases (EC 184.108.40.206) hydrolyse 1,4-β-D-glucosidic linkages, but only 1,3;1,4-β-D-glucans in which the glucosyl residue involved in the glycosidic linkage cleaved is substituted at the C(0)3 position, that is, where the 1,4-β-D-glucosidic linkages are located on the reducing end side of 1,3-β-D-glucosyl residues.
Reaction products released are mainly 1,3;1,4-β-D-tri- and tetrasaccharides (G4G3Gred and G4G4G3Gred), but they also release higher oligosaccharides of up to 10 or more contiguous 1,4-β-D-glucosyl residues with a single reducing terminal 1,3-β-D-glucosyl residue (e.g. G4G4G4G4G4G4G3Gred). These longer oligosaccharides originate from the longer regions of adjacent 1,4-linkages that account for approximately 10% by weight of 1,3;1,4-β-D-glucans in cell walls of the Poaceae .
Kinetics and Mechanism
The stereochemistry of the reaction has been determined experimentally and catalysis by GH17 enzymes occurs via a classical Koshland retaining mechanism with the β-anomeric configuration of the released oligosaccharide being retained . Detailed kinetic analyses are available for three purified barley 1,3-β-D-glucan endohydrolases and two barley 1,3;1,4-β-D-glucan endohydrolases .
Active site labelling with epoxyalkyl β-D-oligoglucoside inhibitors identified Glu231 and Glu232 as the catalytic nucleophiles of the barley 1,3- and 1,3;1,4-β-D-glucan endohydrolases, respectively , located at the bottom of, and about two-thirds of the way along the substrate binding cleft. The general acid/base residue of 1,3;1,4-β-D-glucan endohydrolase was initially identified as Glu288 by chemical labelling procedures [3, 4], but this assignment was subsequently revised and Glu93 was proposed based on primary and tertiary structure similarity of GH17 enzymes with clan GH-A β-glycosidases [5, 6]. The 5-6 Å distance between Glu232 and Glu93 is typical of retaining enzymes.
The crystal structures of the barley 1,3-β-D-glucan endohydrolase isoenzyme GII and 1,3;1,4-β-D-glucan endohydrolase isoenzyme EII have been solved to 2.2-2.3 Å resolution and shown to adopt essentially identical (β/α)8 TIM barrel structures . The rms deviation in Cα positions between the two barley enzymes is 0.65 Å for 278 residues . This indicates that only minor differences in structure and amino acid dispositions at the substrate-binding and catalytic sites are sufficient to change a pre-existing 1,3-β-D-glucan endohydrolase into a highly specific 1,3;1,4-β-D-glucan endohydrolase .
A deep substrate-binding cleft extends across the surface of the enzyme and can accommodate 6-8 glucosyl-binding subsites . The open cleft enables the enzyme to bind at essentially any position along the 1,3;1,4-β-D-glucan substrate and hence to hydrolyse internal glycoside linkages. Like in other clan GH-A structures, the general acid/base and catalytic nucleophile glutamates are positioned on strands β-4 and β-7 [5, 6, 7].
The X-ray crystallographic data provide compelling evidence that the 1,3;1,4-β-D-glucan endohydrolases of barley evolved via the recruitment of pre-existing and widely distributed family GH17 1,3-β-D-glucan endohydrolases . The similarities in substrate specificity between family GH17 and GH16 enzymes has arisen through convergent evolution; the family GH16 enzymes are members of clan-B and have a β-jelly roll structure.
- First sterochemistry determination
- The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley 1,3;1,4-β-glucan by barley 1,3-β-glucanase isoenzyme GII and 1,3;1,4-β-glucanase isoenzyme EII, respectively, was determined by 1H-NMR. Both enzymes catalyze hydrolysis with retention of anomeric configuration (e-->e) and may therefore operate via a double displacement mechanism .
- First catalytic nucleophile identification
- Active site labelling with epoxyalkyl-β-D-oligoglucoside inhibitors identified Glu231 and Glu232 as the catalytic nucleophiles of the barley 1,3- and 1,3;1,4-β-D-glucan endohydrolases, respectively .
- First general acid/base residue identification
- This residue was identified as the conserved Glu residue at the C-terminal end of strand β-4 based on primary and tertiary structure similarity of GH17 enzymes with clan GH-A β-glycosidases [5, 6].
- First 3-D structure
- Barley 1,3-β-D-glucan endohydrolase isoenzyme GII and 1,3;1,4-β-D-glucan endohydrolase isoenzyme EII, solved to 2.2-2.3 Å resolution .
- Woodward, J.R. and Fincher, G.B. (1982) Substrate specificities and kinetic properties of two (1-3),(1-4)-β-D-glucan endo-hydrolases from germinating barley (Hordeum vulgare). Carbohydr. Res. 106, 111-122. DOI:10.1016/S0008-6215(00)80737-5
- Chen L, Sadek M, Stone BA, Brownlee RT, Fincher GB, and Høj PB. Stereochemical course of glucan hydrolysis by barley (1-->3)- and (1-->3, 1-->4)-beta-glucanases. Biochim Biophys Acta. 1995 Nov 15;1253(1):112-6.
- Chen L, Fincher GB, and Høj PB. Evolution of polysaccharide hydrolase substrate specificity. Catalytic amino acids are conserved in barley 1,3-1,4- and 1,3-beta-glucanases. J Biol Chem. 1993 Jun 25;268(18):13318-26.
- Chen L, Garrett TP, Fincher GB, and Høj PB. A tetrad of ionizable amino acids is important for catalysis in barley beta-glucanases. J Biol Chem. 1995 Apr 7;270(14):8093-101.
- Pickersgill R, Harris G, Lo Leggio L, Mayans O, and Jenkins J. Superfamilies: the 4/7 superfamily of beta alpha-barrel glycosidases and the right-handed parallel beta-helix superfamily. Biochem Soc Trans. 1998 May;26(2):190-8.
- Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7090-4.
- Varghese JN, Garrett TP, Colman PM, Chen L, Høj PB, and Fincher GB. Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2785-9.
- Hrmova M and Fincher GB. Structure-function relationships of beta-D-glucan endo- and exohydrolases from higher plants. Plant Mol Biol. 2001 Sep;47(1-2):73-91.