Glycoside Hydrolase Family 28

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• Author: ^^^Richard Pickersgill^^^
• Responsible Curator: ^^^Richard Pickersgill^^^

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Glycoside Hydrolase Family GH28
Clan	GH-N
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH28.html

Substrate specificities

The overwhelming majority of enzymes in this family are polygalacturonases. They hydrolyse the alpha-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 alpha-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 [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

Family GH28 enzymes are inverting enzymes; they harness a single displacement mechanism as revealed by 1H-NMR spectroscopy of the products of hydrolysis in D2O reaction mixtures [6]. Subsequently, the rhamnogalacturonases were also shown to invert the configuration of the newly formed reducing end of the polysaccharide [4].

Catalytic Residues

The crystal structure of rhamnogalacturonase revealed the cluster of aspartates involved in catalysis [7]. Pickersgill et al [8] realised that protonation of the glycosidic oxygen and nucleophilic attack at the anomeric carbon may be from the same side of the bond in alpha-linked polysaccharides rather than opposite sides with a resulting shorter separation of carboxylates than standard for cleaving substrates with beta-linkages explaining the short spacing between the conserved carboxylates in the GH28 hydrolases. The clearest assignment of the catalytic residues comes from the work of van Santen et al [9]; Asp201 is proposed to act as the general acid (proton donor), while Asp180 and Asp202 active the catalytic water molecule (numbers are given for Aspergillus niger polygalacturonase).

Three-dimensional structures

Rhamnogalacturonase (RGase A) from Aspergillus aculeatus [7], endo-polygalacturonase from Erwinia carotovora [8], endo-polygalacturonase II from Aspergillus ? [9], endogalacturonase from ? [10], endo-polygalacturonase I from Aspergillus ? [11], exogalacturonase from ? [12].

Family Firsts

First sterochemistry determination

Endopolygalacturonases from Aspergillus niger and Aspergillus tubingensis [6].

First catalytic acid identification

van Santen et al [9]; Asp201 (197) is proposed to act as the catalytic acid (proton donor), while Asp180 (180) and Asp202 (198) active the catalytic water molecule (numbers are given for the Aspergillus niger and Erwinia carotavora residues in parentheses).

First 3-D structure

Rhamnogalacturonase (RGase-A) from Aspergillus aculeatus [7]. First polygalacturonase structure: Erwinia carotavora polygalacturonase [8].

First complexes

Product complex (+1 subsite) and a complex including a furanose isomer (-1) [10]. A product complex in an exo-polygalacturonase illuminates the structural basis for its exo-activity [11].

References

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