Glycoside Hydrolase Family 13

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• Authors: ^^^Birte Svensson^^^ and ^^^Stefan Janecek^^^
• Responsible Curator: ^^^Birte Svensson^^^

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Glycoside Hydrolase Family GH13
Clan	GH-H
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH13.html

Substrate specificities

Family GH13 is the major glycoside hydrolase family acting on α-glucoside containing substrates. It has recently been subdivided into 35 subfamilies [1], currently 36 subfamilies are given in CAZy [2]. There has been a number of reviews concerned with α-amylases [3, 4, 5, 6]. GH13 contains hydrolases, transglycosidases and isomerases, noticeably amino acid transporters [7], which have no glycoside activity, are GH13 members. The enzymes are found in a very wide range of organisms from all kingdoms. While known specificities are indicated by the enzyme named as follows below, for several of these enzymes numerous have been characterized to comprise subspecificities defined by structural requirements for preferred substrates or the structure of the predominant product(s). Known enzymes currently include: α-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41); cyclomaltodextrin glucanotransferase (EC 2.4.1.19); cyclomaltodextrinase (EC 3.2.1.54); trehalose-6-phosphate hydrolase (EC 3.2.1.93); oligo-α-glucosidase (EC 3.2.1.10); maltogenic amylase (EC 3.2.1.133); neopullulanase (EC 3.2.1.135); α-glucosidase (EC 3.2.1.20); maltotetraose-forming α-amylase (EC 3.2.1.60); isoamylase (EC 3.2.1.68); glucodextranase (EC 3.2.1.70); maltohexaose-forming α-amylase (EC 3.2.1.98); maltotriose-forming α-amylase (EC 3.2.1.116); branching enzyme (EC 2.4.1.18); trehalose synthase (EC 5.4.99.16); 4-α-glucanotransferase (EC 2.4.1.25); maltopentaose-forming α-amylase (EC 3.2.1.-); amylosucrase (EC 2.4.1.4); sucrose phosphorylase (EC 2.4.1.7); malto-oligosyltrehalose trehalohydrolase (EC 3.2.1.141); isomaltulose synthase (EC 5.4.99.11); amino acid transporter . Interestingly several members of GH13 contains carbohydrate binding modules (CBMs) referred to as starch binding domains, and belonging to CBM20, 21, 25, 26, 34, 41, 45, 48, 53, and 58 [8, 9, 10, 11, 12].

The different enzymes have a wide range of different preferred substrates and product. For example, the α-amylases prefer polysaccharides of the α-(1,4)-glucan type such as amylose and also amylopectin, but they do attack also the supramolecular structures represented by starch granules and glycogen particles and have some significant albeit slower turn-over of maltooligosaccharides of a certain degree of polymerization. These preferred substrate profiles can be manipulated through protein engineering.

Kinetics and Mechanism

GH Family 13 enzymes are retaining as was first demonstrated by quantitative gas liquid chromatogrphic analysis of formation of a-maltose fro diferent maltosides (Kimura and Chiba, 1983) futher supported y NMR analysis of the release of a-maltose from similar substrates (isoda et al 1992) as demosntrated for a number of different a-amylasesref) and they follow the classical Koshland double-displacement mechanism (ref). This has been supported by covalent labeling using 4-deoxy-maltotriose-fluoride labelling the catalytic nucleophile (Uitdehaag et al., 1999), numerous three-dimensional structures (ref), and site-directed mutational substitution of the catalytic site residues (ref).

Some of the Family 13 members use a multiple attack or processive mechanism (refs) involving several glycoside bond cleavages to be executed in the same enzyme-substrate encounter.

In several cases has the binding energies been determined using subsite mapping (refs) which give a typical subsite binding energy profile for individual enzymes (ref).

Several α-amylases have been reported to interact with polymeric substrates at surface sites situated as a certain distance of the active site (ref).

Finally interaction with insoluble substrates such as starch granules or glycogen can occur both at these sites (ref) as well as by the involvement of separate binding modules referred to as starch binding domains (ref).

Catalytic Residues

The catalytic residues have been identified from early crystal structures (ref). In fact throughout the Family 13 only three residues are totally conserved (except for in the amino acid transporters) these include an Asp catalytic nucleophile, a Glu general acid/base, and a catalytic site residue which is an Asp that participates critically in stabilizing the transition state (ref). Numerous mutational analyses have been performed to confirm the essential roles of these three residues in catalysis, and normally the loss in activity is four-five orders of magnitude.

Three-dimensional structures

Numerous GH13 subfamilies contain members for which a three-dimensional structure has been determined. The first crystal are reported for barley α-amylase were reported in the mid-forties, however the first crystal structures were of porcine pancreatic and α-amylase and TAKA-amylase (ref). This was followed by structures of other α-amylases from bacteria and from higher plants (refs) and the industrially important cyclodextrin glucanotransferase (ref). Later on the amylopectin debranching isoamylase and the related pullulanases were structure determined (ref). More recently amylosucrase (ref), an exo-dextranase (ref) and also a dextrinsucrase (ref) was solved. Among the solved structures are numerous site-directed mutant and numerous ligand complexed forms. There are structurals available for many of these specificities, but some still remain to be determined.

Family Firsts

First sterochemistry determination

α-Maltose was released from different α-maltosides by B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, as determined by quantitative gas liquid chromatography [13]. This was as well demonstrated by NMR analysis of the anomeric configuration of the released product [14].

First catalytic nucleophile

A b-glycosidic covalent bond was formed in the intermediate of mechanism between the catalytic nucleophile (D229) of Bacillus circulans 251 CGTase and a maltotriosyl moiety [15]. Mutational analysis of human pancreatic α-amylase provided strong support for D197 being the catalytic nucleophile as demonstrated by kinetics analysis [16].

First general acid/base

Mutatitional analysis of human pancreatic α-amylase using enzymatic kinetics and structural analysis provided strong support for E233 playing the role of the catalytic acid/base [16].

First 3-D structure

The first high-resolution three-dimensional structure was determined for Taka-amylase A [17].

Proteinaceous inhibitors Exogenous and endogenous inhibitory protein have been reported from microorganisms and plants (ref) directed towards α-amylases (ref) and limit dextrinases (ref).

References

1. Stam MR, Danchin EG, Rancurel C, Coutinho PM, and Henrissat B. (2006). Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins. Protein Eng Des Sel. 2006;19(12):555-62. DOI:10.1093/protein/gzl044 |
2. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
3. Svensson B (1994). Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol. 1994;25(2):141-57. DOI:10.1007/BF00023233 |
4. Janecek S (1997). alpha-Amylase family: molecular biology and evolution. Prog Biophys Mol Biol. 1997;67(1):67-97. DOI:10.1016/s0079-6107(97)00015-1 |
5. Kuriki T and Imanaka T. (1999). The concept of the alpha-amylase family: structural similarity and common catalytic mechanism. J Biosci Bioeng. 1999;87(5):557-65. DOI:10.1016/s1389-1723(99)80114-5 |
6. MacGregor EA, Janecek S, and Svensson B. (2001). Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta. 2001;1546(1):1-20. DOI:10.1016/s0167-4838(00)00302-2 |
7. Janecek S, Svensson B, and Henrissat B. (1997). Domain evolution in the alpha-amylase family. J Mol Evol. 1997;45(3):322-31. DOI:10.1007/pl00006236 |
9. Janecek S and Sevcík J. (1999). The evolution of starch-binding domain. FEBS Lett. 1999;456(1):119-25. DOI:10.1016/s0014-5793(99)00919-9 |
10. Rodríguez-Sanoja R, Oviedo N, and Sánchez S. (2005). Microbial starch-binding domain. Curr Opin Microbiol. 2005;8(3):260-7. DOI:10.1016/j.mib.2005.04.013 |
11. Machovic M and Janecek S. (2006). Starch-binding domains in the post-genome era. Cell Mol Life Sci. 2006;63(23):2710-24. DOI:10.1007/s00018-006-6246-9 |
12. Christiansen C, Abou Hachem M, Janecek S, Viksø-Nielsen A, Blennow A, and Svensson B. (2009). The carbohydrate-binding module family 20--diversity, structure, and function. FEBS J. 2009;276(18):5006-29. DOI:10.1111/j.1742-4658.2009.07221.x |
18. Svensson B, Jespersen H, Sierks MR, and MacGregor EA. (1989). Sequence homology between putative raw-starch binding domains from different starch-degrading enzymes. Biochem J. 1989;264(1):309-11. DOI:10.1042/bj2640309 |