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Glycoside Hydrolase Family 28
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|Glycoside Hydrolase Family GH28|
|Active site residues||known|
|CAZy DB link|
The overwhelming majority of glycoside hydrolases of this family are polygalacturonases. They hydrolyse the α-1,4 glycosidic linkage between galacturonate residues in polygalacturonic acid. Both endo and exo acting polygalacturonases are represented. Polygalacturonic acid, with varying degrees of C6 methylation and acetylation, forms the smooth homogalacturonan region of pectin. There are also some enzymes in this family active against rhamnogalacturonan which forms the branched part of the pectin molecule. Rhamnogalacturonases cleave the α-1,2 linkage between galacturonic acid and rhamnose residues. Two other enzymes rhamnohydrolase and rhamnogalacturonan galacturonohydrolase cleave off single terminal carbohydrate units, rhamnose and galacturonate respectively, from the non-reducing end of rhamnogalacturonan .
Kinetics and Mechanism
Family GH28 enzymes follow an inverting mechanism; they harness a single displacement mechanism as revealed by 1H-NMR spectroscopy of the products of hydrolysis in D2O reaction mixtures . Subsequently, the rhamnogalacturonases were also shown to invert the configuration of the newly formed reducing end of the polysaccharide .
The crystal structure of rhamnogalacturonase revealed a cluster of aspartates involved in catalysis . It was subsequently realised that protonation of the glycosidic oxygen and nucleophilic attack at the anomeric carbon may be from the same side of the bond in α-linked polysaccharides rather than opposite sides with a resulting shorter separation of carboxylates than standard for cleaving substrates with β-linkages explaining the short spacing between the conserved carboxylates in the GH28 hydrolases . These authors identified Asp202, Asp223 and Asp224 as the catalytic residues. The clearest assignment of the role of the catalytic residues comes from the work of van Santen et al  based on the results of mutagenesis and comparison with phage 22 tailspike protein . Asp201 (Asp223) is proposed to act as the general acid residue (proton donor), whereas Asp180 (Asp202) and Asp202 (Asp224) are general bases that activate the nucleophilic water molecule (numbers are given for Aspergillus niger and in parantheses for Ewinia carotovora polygalacturonase).
The structure of rhamnogalacturonase (RGase A) from Aspergillus aculeatus  revealed the signature parallel β-helix architecture common to several pectin active enzymes including family 1 pectate lyases (PL1). The GH28 enzymes are distinguished from the lyases by having four, not three, parallel β-sheets extending along their longitudinal axes. Compared to rhamnogalacturonase, polygalacturonase lacks the C-terminal turn of β-helix having ten turns, not eleven . The structure of exopolygalacturonase from Yersinia enterocolitica shows how amino acid inserts close off the open substrate binding cleft of endopolygalacturonase to form an exopolygalacturonase .
- First sterochemistry determination
- Endopolygalacturonases from Aspergillus niger and Aspergillus tubingensis .
- First general acid identification
- Aspergillus niger endopolygalacturonase. Asp201 (223) is proposed to act as the catalytic acid (proton donor). Nnumbers are given for the Aspergillus niger and in parentheses forErwinia carotovora polygalacturonase) .
- First general base identification
- Aspergillus niger endopolygalacturonase. Asp180 (202) and Asp202 (224) active the nucleophilic water molecule (numbers are given for the Aspergillus niger and in parentheses for Erwinia carotovora polygalacturonase) .
- First 3-D structure
- Rhamnogalacturonase (RGase-A) from Aspergillus aculeatus . First polygalacturonase structure, Erwinia carotovora polygalacturonase .
- First complexes
- Product complex (+1 subsite) and a complex including a furanose isomer (-1) . A product complex in an exo-polygalacturonase illuminates the structural basis for its exo-activity .
- J Visser, Ir and A. G. J. Voragen. (1996) Pectins and Pectinases. Elsevier.
- Biely P, Benen J, Heinrichová K, Kester HC, and Visser J. (1996). Inversion of configuration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases. FEBS Lett. 1996;382(3):249-55. DOI:10.1016/0014-5793(96)00171-8 |
- Pitson SM, Mutter M, van den Broek LA, Voragen AG, and Beldman G. (1998). Stereochemical course of hydrolysis catalysed by alpha-L-rhamnosyl and alpha-D-galacturonosyl hydrolases from Aspergillus aculeatus. Biochem Biophys Res Commun. 1998;242(3):552-9. DOI:10.1006/bbrc.1997.8009 |
- Petersen TN, Kauppinen S, and Larsen S. (1997). The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. Structure. 1997;5(4):533-44. DOI:10.1016/s0969-2126(97)00209-8 |
- Pickersgill R, Smith D, Worboys K, and Jenkins J. (1998). Crystal structure of polygalacturonase from Erwinia carotovora ssp. carotovora. J Biol Chem. 1998;273(38):24660-4. DOI:10.1074/jbc.273.38.24660 |
- van Santen Y, Benen JA, Schröter KH, Kalk KH, Armand S, Visser J, and Dijkstra BW. (1999). 1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. J Biol Chem. 1999;274(43):30474-80. DOI:10.1074/jbc.274.43.30474 |
- Steinbacher S, Miller S, Baxa U, Budisa N, Weintraub A, Seckler R, and Huber R. (1997). Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage. J Mol Biol. 1997;267(4):865-80. DOI:10.1006/jmbi.1997.0922 |
- Abbott DW and Boraston AB. (2007). The structural basis for exopolygalacturonase activity in a family 28 glycoside hydrolase. J Mol Biol. 2007;368(5):1215-22. DOI:10.1016/j.jmb.2007.02.083 |
- Shimizu T, Nakatsu T, Miyairi K, Okuno T, and Kato H. (2002). Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution. Biochemistry. 2002;41(21):6651-9. DOI:10.1021/bi025541a |