Glycoside Hydrolase Family 17

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

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/fam/GHnn.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)-beta-D-glucan endohydrolases (EC 3.1.2.39) and (1,3;1,4)-beta-D-glucan endohydrolases (EC 3.1.2.73). A (1,3)-beta-D-glucan exohydrolase (EC 3.1.2.58) is also classified in this family. The family 17 enzymes have quite distinct amino acid sequences and 3D structures compared with the (1,3)-beta-D-glucan endohydrolases and (1,3;1,4)-beta-D-glucan endohydrolases that have similar substrate specificities but are classified in families GH16, GH55, GH64 and GH81.

The family GH17 (1,3)-beta-D-glucan endohydrolases hydrolyse internal (1,3)-beta-D-glucosidic linkages in polysaccharides, but usually require a region of contiguous unbranched, un-substituted (1,3)-beta-D-glucosyl residues for activity. The enzymes release (1,3)-beta-D-oligoglucosides of DP 2-5 as their major products. Because the (1,3)-beta-D-glucan endohydrolases require a region of contiguous unbranched, un-substituted (1,3)-beta-D-glucosyl residues for activity, they are unable to hydrolyse the single (1,3)-beta-D-glucosidic linkages in (1,3;1,4)-beta-D-glucans from the Poaceae, but they will hydrolyse (1,3)-beta-D-glucosidic linkages in fungal (1,3;1,6)-beta-D-glucans, provided an appropriate region of contiguous un-substituted (1,3)-beta-D-glucosyl residues is available. The family GH17 (1,3;1,4)-beta-D-glucan endohydrolases (EC 3.1.2.73) hydrolyse (1,4)-beta-D-glucosidic linkages, but only (1,3;1,4)-beta-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)-beta-D-glucosidic linkages are located on the reducing end side of (1,3)-beta-D-glucosyl residues.

Reaction products released are mainly (1,3;1,4)-beta-D-tri- and tetrasaccharides (G4G3G_red and G4G4G3G_red), but they also release higher oligosaccharides of up to 10 or more contiguous (1,4)-beta-D-glucosyl residues with a single reducing terminal (1,3)-beta-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)-beta-D-glucans in cell walls of the Poaceae [1].

This is an example of how to make references to a journal article [2]. (See the References section below). Multiple references can go in the same place like this [2, 3]. You can even cite books using just the ISBN [4]. References that are not in PubMed can be typed in by hand [5].

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 beta-anomeric configuration of the released oligosaccharide is retained [6]. Detailed kinetic analyses are available for three purified barley (1,3)-β-d-glucan endohydrolases and two barley (1,3;1,4)-beta-D-glucan endohydrolases [6].

Catalytic Residues

Active site labelling with epoxyalkyl-beta-D-oligoglucoside inhibitors identified Glu231 and Glu232 as the catalytic nucleophiles of the barley (1,3)- and (1,3;1,4)-beta-D-glucan endohydrolases, respectively [7], 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)-beta-D-glucan endohydrolase was initially identified as Glu288 by chemical labelling procedures [7, 8], but this assignment was subsequently revised and Glu93 was proposed based on primary and tertiary structural similarity of GH17 enzymes with clan GH-A beta-glycosidases [9, 10]. The 5-6 Å distance between Glu232 and Glu93 is more typical of retaining enzymes.

Three-dimensional structures

The crystal structures of the barley (1,3)-beta-D-glucan endohydrolase isoenzyme GII and (1,3;1,4)-beta-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 [11]. The rms deviation in Cα positions between the two barley enzymes is 0.65 Å for 278 residues [8].

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

The polypeptide backbones of the two enzymes are almost perfectly superimposable, and this indicates that only minor differences in structure and amino acid dispositions at the substrate-binding and catalytic sites are necessary to change a pre-existing (1,3)-β-d-glucan endohydrolase into a highly specific (1,3;1,4)-beta-D-glucan endohydrolase [8]. The location of the catalytic apparatus is conserved in these enzymes with the catalytic acid/base and nucleophile glutamates positioned on strands β-4 and β-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 specificities between the the family 17 and family 16 enzymes has arisen through convergent evolution; the family 16 enzymes are members of clan-B and have a β-jelly roll conformation.

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Family Firsts

First sterochemistry determination

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

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [5].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [3].

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [4].

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

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. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 |

5. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006

6. Chen L, Sadek M, Stone BA, Brownlee RTC, Fincher GB and Høj PB (1995) Stereochemical course of glucan hydrolysis by barley 1,3- and 1,3;1,4-beta-glucan endohydrolases. Biochim. Biophys. Acta 1253, 112-116

7. 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. 268, 13318-13326

8. Chen L, Garrett TPJ, Fincher GB and Høj PB (1995) A tetrad of ionizable amino acids is important for catalysis in barley ß-glucanases. J. Biol. Chem. 270, 8093-8101

9. 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 |
10. 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 |
11. 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 |