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 13 is the major glycoside hydrolase family acting on α-glucoside containing substrates. It has recently been subdivided into subfamilies (Stam et al., 2005). There has been a number of reviews concerned with a-amylases (ref). GH13 contains hydrolases, transglycosidases and isomerases, noticeably amino acid transporters, which have no glycoside activity, are GH13 members. The enzymes are found in a very wide range of organisms form all kingdoms. While known specificities are indicated by the enzyme named as follow 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 (refs).

The different enzymes have a wide range of different preferred substrates and product. E.g. the α-amylases prefer polysaccharides of the α(1,4)-glucan type such as amylase and also amylopectin, but they do attack also the supramolecular structures represented by starch granules and glycogen particles and have some significant. Albeit lower turn-over of maltooligosaccharides of a certain degree of polymerization. These preferred substrate profiles can be manipulated through protein engineering. Content is to be added here.

This is an example of how to make references to a journal article [1]. (See the References section below). Multiple references can go in the same place like this [1, 2]. You can even cite books using just the ISBN [3]. References that are not in PubMed can be typed in by hand [4].

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 a-amylase,as determined by quantitative gas liquid chromatography (Kimura and Chiba, abc 1983). This ws as well demonstrated by nmr analysis of the anomeric configuration of the released product (Isoda et al., 1992, J biochem(Cite some reference here, with a short (1-2 sentence) explanation [1].

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 (Uitdehaag et al. Natur structual biology 1999). Mutational analysis of human pancreatic a-amylase provided strong support for D197 being the catalytic nucleophile as demonstrated by kinetics analysis (Rydberg et al, Biochemistry 2002). T Cite some reference here, with a short (1-2 sentence) explanation [4].

First general acid/base Mutatitional analysis of human pancreatic a-amylase using enzymatic kinetics and structural analysis provided stron support for E233 playing the role of the catalytic acid&base (Rydberg et al., Biochemistry 2002)

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

First 3-D structure

the first high/resolution threedimensional structure was determined for Taka/aylase A (Matsuura et al., j biochem. 1984)Cite some reference here, with a short (1-2 sentence) explanation [3].

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. 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 |
2. 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 |

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