Glycoside Hydrolase Family 18

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• Author: ^^^Gideon Davies^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH18
Clan	GH-K
Mechanism	retaining
Active site residues	known (acid/neighbouring group)
CAZy DB link
http://www.cazy.org/fam/GH18.html

Substrate specificities

GH18 is unusual in having both catalytically active chitinase (EC 3.2.1.14) and endo-β-N-acetylglucosaminidases (EC 3.2.1.96) but there are also sub-families of non-hydrolytic proteins that function as carbohydrate binding modules / "lectins" or as xylanase inhibitors.

Kinetics and Mechanism

GH18 enzymes belong to a growing group of enzymes (now including GH families 18, 20,25, 56, 84, and 85) that perform a double-displacement reaction but instead of the more common enzyme-derived nucleophile they utlize the N-acetamido carbonyl oxygen in what is termed "neighbouring group participation" / "substrate participation" or "anchimeric assistance". Figures showing such a mechanism date back to Koshland's 1953 review [1], indeed they frequent the chemical literature of participating groups long before that, but it is primarily through the work on GH18 [2] and soon after GH20 [3, 4] that such a mechanism became well established. In such a mechanism, which occurs with (net) retention of anomeric configuration, the enzyme provides a catalytic acid function to protonate the leaving group to facilitate its departure with the substrate carbonyl oxygen playing the role of nucleophile to generate a bicyclic "oxazoline" intermediate (which subsequently breaks down following the microscopic reverse via hydrolysis or occasionally transglycosylation). Such a mechanism has a number of facets, one of which is its potential inhibition using thiazolines [5].

Catalytic Residues

The catalytically active GH18 enzymes use a double displacement reaction mechanism with "neighbouring group participation". Hence there is a catalytic acid residue (glutamate in family GH18, but often also Asp in other families using this mechanism) and in all families apart from GH85 (where this residue is an amide), a second carboxylate (here Asp) acts to deprotonate the N-acetamido nitrogen during oxazoline formation/breakdown. In family GH18 the two catalytic carboxylates are found in an D-X-G motif whereas in some families the carboxylates may be adjacent such as the DD motif in family GH84 (for example see [6]). The physical separation of the two catalytic residues (with the second not in a position to act as a nucleophile itself) has led to confusion in some literature that GH18 and other enzymes (notably GH25) may be inverting enzymes; this is certainly not the case for GH18 and is unlikely to be the case for GH25.

Three-dimensional structures

Although these enzymes are frequently multi-modular, the catalytic domains are α / β barrels [7, 8].

Work on the conformational itinerary of catalysis which is extremely similar to other retaining enzymes active on gluco-configured substrates, was provided by the van Aalten group [9] in 2001 through the trapping of a distorted Michaelis complex in ^1,4B conformation and thus extremely similar to the ¹S₃ skew boats oberserved in GH5 [10] for example or the ⁴E conformation originally seen for a "neighboring group" enzyme in GH20 cite>Tews1996. More recetly, a similar conformation has been observed for the Michaelis complex of another neighboring group enzymem the GH84 O-GlcNAcase [6]. Fungal GH18 enzymes are considered as possible therapeutic targets and a number of programmes are probing this area (for example [11]).

One unusual feauture of GH18 are the latge numbers of sequences that encode catalytically-inactive proteins that function as enzyme inhibitors or lectins. SOMEONE ELSE WRITE THIS?

Daan Chilectin paper [12] Hennig Concanavalin B paper [13] Narbonin [14]

Review of non-catalytic GH18 as enzyme inhibitors [15].

Family Firsts

First sterochemistry determination

Sometimes incorrectly reported as inverting, this family performs catalysis with retention of anomeric configuration as first shown on the Bacillus ciculans enzyme [16].

First catalytic nucleophile identification

This family is one of many that uses neighbouring group participation for catalysis with the N-acetyl carbonyl group acting as the nucleophile; first proposed (I believe) for this family in [2].

First general acid/base residue identification

On the basis of 3-D structure [7].

First 3-D structure

The first two 3-D structures for catalytically active GH18 members were the Serratia marcescens chitinase A and the plant defence protein hevamine published "back-to-back" in Structure in 1994 [7, 8]. In retrospect, however, the non-catalytic "Narbonin" structure was arguably the first GH18 3-D structure, althoigh it is has no enzymatic activity [14].

References

2. Terwisscha van Scheltinga AC, Armand S, Kalk KH, Isogai A, Henrissat B, and Dijkstra BW. (1995). Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Biochemistry. 1995;34(48):15619-23. DOI:10.1021/bi00048a003 |
3. Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, and Vorgias CE. (1996). Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nat Struct Biol. 1996;3(7):638-48. DOI:10.1038/nsb0796-638 |
4. Drouillard S, Armand S, Davies GJ, Vorgias CE, and Henrissat B. (1997). Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. Biochem J. 1997;328 ( Pt 3)(Pt 3):945-9. DOI:10.1042/bj3280945 |
5. Macdonald JM, Tarling CA, Taylor EJ, Dennis RJ, Myers DS, Knapp S, Davies GJ, and Withers SG. (2010). Chitinase inhibition by chitobiose and chitotriose thiazolines. Angew Chem Int Ed Engl. 2010;49(14):2599-602. DOI:10.1002/anie.200906644 |
6. He Y, Macauley MS, Stubbs KA, Vocadlo DJ, and Davies GJ. (2010). Visualizing the reaction coordinate of an O-GlcNAc hydrolase. J Am Chem Soc. 2010;132(6):1807-9. DOI:10.1021/ja9086769 |
7. Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, and Vorgias CE. (1994). Crystal structure of a bacterial chitinase at 2.3 A resolution. Structure. 1994;2(12):1169-80. DOI:10.1016/s0969-2126(94)00119-7 |
9. van Aalten DM, Komander D, Synstad B, Gåseidnes S, Peter MG, and Eijsink VG. (2001). Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci U S A. 2001;98(16):8979-84. DOI:10.1073/pnas.151103798 |
10. Davies GJ, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Schülein M, and Withers SG. (1998). Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase. Biochemistry. 1998;37(34):11707-13. DOI:10.1021/bi981315i |
11. Houston DR, Shiomi K, Arai N, Omura S, Peter MG, Turberg A, Synstad B, Eijsink VG, and van Aalten DM. (2002). High-resolution structures of a chitinase complexed with natural product cyclopentapeptide inhibitors: mimicry of carbohydrate substrate. Proc Natl Acad Sci U S A. 2002;99(14):9127-32. DOI:10.1073/pnas.132060599 |
12. Houston DR, Recklies AD, Krupa JC, and van Aalten DM. (2003). Structure and ligand-induced conformational change of the 39-kDa glycoprotein from human articular chondrocytes. J Biol Chem. 2003;278(32):30206-12. DOI:10.1074/jbc.M303371200 |
13. Hennig M, Jansonius JN, Terwisscha van Scheltinga AC, Dijkstra BW, and Schlesier B. (1995). Crystal structure of concanavalin B at 1.65 A resolution. An "inactivated" chitinase from seeds of Canavalia ensiformis. J Mol Biol. 1995;254(2):237-46. DOI:10.1006/jmbi.1995.0614 |
14. Hennig M, Schlesier B, Dauter Z, Pfeffer S, Betzel C, Höhne WE, and Wilson KS. (1992). A TIM barrel protein without enzymatic activity? Crystal-structure of narbonin at 1.8 A resolution. FEBS Lett. 1992;306(1):80-4. DOI:10.1016/0014-5793(92)80842-5 |
15. Durand A, Hughes R, Roussel A, Flatman R, Henrissat B, and Juge N. (2005). Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18. FEBS J. 2005;272(7):1745-55. DOI:10.1111/j.1742-4658.2005.04606.x |
16. Armand S, Tomita H, Heyraud A, Gey C, Watanabe T, and Henrissat B. (1994). Stereochemical course of the hydrolysis reaction catalyzed by chitinases A1 and D from Bacillus circulans WL-12. FEBS Lett. 1994;343(2):177-80. DOI:10.1016/0014-5793(94)80314-5 |
17. Terwisscha van Scheltinga AC, Kalk KH, Beintema JJ, and Dijkstra BW. (1994). Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure. 1994;2(12):1181-9. DOI:10.1016/s0969-2126(94)00120-0 |