Glycoside Hydrolase Family 17

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• Author: ^^^Geoff Fincher^^^
• Responsible Curator: ^^^Bernard Henrissat^^^

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Glycoside Hydrolase Family GH17
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH17.html

Substrate specificities

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 3.2.1.39) and 1,3;1,4-β-D-glucan endohydrolases (EC 3.1.2.73). A 1,3-β-D-glucan exohydrolase (EC 3.1.2.58) 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, un-substituted 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 3.1.2.73) 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 (G4G3G_red and G4G4G3G_red), 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. G4G4G4G4G4G4G3G_red). 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 [1].

Kinetics and Mechanism

The stereochemistry of the reaction has been determined experimentally and catalysis by GH17 enzymes occurs via a double displacement mechanism and the β-anomeric configuration of the released oligosaccharide is retained [2]. 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 [2].

Catalytic Residues

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 [3], located at the bottom of, and about two-thirds of the way along the substrate binding cleft. The catalytic 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.

Three-dimensional structures

Superposition of the polypeptide backbones of the barley (1,3)-β-D-glucan endohydrolase isoenzyme GII (green) and (1,3;1,4)-β-D-glucan endohydrolase isoenzyme EII (yellow). From [7]

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 [7]. The rms deviation in Cα positions between the two barley enzymes is 0.65 Å for 278 residues [7]. 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 [7].

A deep substrate-binding cleft extends across the surface of the enzyme and can accommodate 6-8 glucosyl-binding subsites [7]. 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 catalytic acid/base and 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 [6]. 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.

Family Firsts

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 [2].

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 [3].

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

Cite some reference here, with a short (1-2 sentence) explanation [7].

References

1. 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

2. Chen L, Sadek M, Stone BA, Brownlee RT, Fincher GB, and Høj PB. (1995). Stereochemical course of glucan hydrolysis by barley (1-->3)- and (1-->3, 1-->4)-beta-glucanases. Biochim Biophys Acta. 1995;1253(1):112-6. DOI:10.1016/0167-4838(95)00157-p |
3. Chen L, Fincher GB, and Høj PB. (1993). 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;268(18):13318-26. | Google Books | Open Library
4. Chen L, Garrett TP, Fincher GB, and Høj PB. (1995). A tetrad of ionizable amino acids is important for catalysis in barley beta-glucanases. J Biol Chem. 1995;270(14):8093-101. DOI:10.1074/jbc.270.14.8093 |
5. Pickersgill R, Harris G, Lo Leggio L, Mayans O, and Jenkins J. (1998). Superfamilies: the 4/7 superfamily of beta alpha-barrel glycosidases and the right-handed parallel beta-helix superfamily. Biochem Soc Trans. 1998;26(2):190-8. DOI:10.1042/bst0260190 |
6. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 |
7. Varghese JN, Garrett TP, Colman PM, Chen L, Høj PB, and Fincher GB. (1994). Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities. Proc Natl Acad Sci U S A. 1994;91(7):2785-9. DOI:10.1073/pnas.91.7.2785 |
8. Hrmova M and Fincher GB. (2001). Structure-function relationships of beta-D-glucan endo- and exohydrolases from higher plants. Plant Mol Biol. 2001;47(1-2):73-91. | Google Books | Open Library