Difference between revisions of "Glycoside Hydrolase Family 13"

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

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 substrates containing α-glucoside linkages. A number of reviews are concerned with α-amylases [1, 2, 3, 4, 5]. GH13 contains hydrolases, transglycosidases and isomerases [4]; noticeably animal amino acid transporters [6], which have no glycosidase activity [7], are also 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 listed below, for several of these numerous enzymes are characterized representing subspecificities defined by structural requirements for preferred substrates or the structure of the predominant product(s). Described enzymes include: α-amylase (EC 3.2.1.1); oligo-1,6-glucosidase (EC 3.2.1.10); α-glucosidase (EC 3.2.1.20); pullulanase (EC 3.2.1.41); cyclomaltodextrinase (EC 3.2.1.54); maltotetraose-forming α-amylase (EC 3.2.1.60); isoamylase (EC 3.2.1.68); dextran glucosidase (EC 3.2.1.70); trehalose-6-phosphate hydrolase (EC 3.2.1.93); maltohexaose-forming α-amylase (EC 3.2.1.98); maltotriose-forming α-amylase (EC 3.2.1.116); maltogenic amylase (EC 3.2.1.133); neopullulanase (EC 3.2.1.135); malto-oligosyltrehalose trehalohydrolase (EC 3.2.1.141); maltopentaose-forming α-amylase (EC 3.2.1.-); amylosucrase (EC 2.4.1.4); sucrose phosphorylase (EC 2.4.1.7); branching enzyme (EC 2.4.1.18); cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19); 4-α-glucanotransferase (EC 2.4.1.25); isomaltulose synthase (EC 5.4.99.11); trehalose synthase (EC 5.4.99.16). As mentioned above, heavy-chains of heteromeric amino acid transporters belong to the GH13 [6, 8]. Among thousands of sequences and ~30 different enzymes specificities [9] many are closely related to each other, GH13 therefore has officially been subdivided into almost 40 subfamilies [10]; several subfamilies, e.g., the oligo-1,6-glucosidase and neopullulanase subfamilies were described earlier [11]. Noticeably a considerable number of GH13 members contain carbohydrate binding modules (CBMs) referred to as starch binding domains belonging to CBM20, 21, 25, 26, 34, 41, 45, 48, 53, and 58 [12, 13, 14, 15, 16].

The GH13 enzymes have a wide range of different preferred substrates and products. For example, the α-amylases prefer polysaccharides of the α-1,4-glucan type, such as amylose and amylopectin, but are also able to attack the supramolecular structures represented by starch granules and glycogen particles. Besides they have some significant albeit slower turn-over of maltooligosaccharides of a certain degree of polymerization. These typical substrate profiles can be manipulated through protein engineering.

The α-amylase family was defined in 1991 as family GH13 when the sequence-based classification of glycoside hydrolases was created [17]. The α-amylase family as an enzyme family, however, was established based on results of several independent findings focused on starch hydrolases and related enzymes [18, 19, 20, 21]. These enzymes were shown to exhibit sequence similarities and, at that time, a predicted (β/α)8-barrel (i.e. TIM-barrel) fold. The basic criteria for a protein to be a member of the α-amylase family were as follows [21]: the enzyme should (i) act on the α-glucosidic linkages; (ii) hydrolyse or form by transglycosylation the α-glucosidic linkages; (iii) contain the four conserved sequence regions in its amino acid sequence; and (iv) possess the catalytic triad formed by the three residues corresponding to Asp206, Glu230 and Asp297 of Taka-amylase A (the α-amylase from Aspergillus oryzae). A dramatic increase of the number of GH13 members to several thousands [9] offered a greater variety in both substrate and product specificities and sequence diversity so that the above criteria had to be updated. For example, also enzymes active on α-1,1-, α-1,2-, α-1,3- and α-1,5-glucosidic linkages belong to the α-amylase family [4] and the four best known and well-accepted conserved sequence regions, defined first for eleven α-amylases [22], were completed by the additional three regions [23, 24] which can often help to assign the correct enzyme specificity of α-amylase family members (for a review, see [25]). Of note may be the enzyme neopullulanase [26] that was found to catalyze both the hydrolysis of α-1,4- and α-1,6-glucosidic bonds as well as the transglycosylation to form these two types of glucosidic bonds.

The α-amylase family represents a clan GH-H of three glycoside hydrolase families GH13, GH70 and GH77 [4], and should be distinguished from the second smaller α-amylase family GH57 [27]. A remote homology to the family GH31 has also been discussed [28].

The evolutionary relationships were described for the entire GH13 family [2, 10, 20, 29] and some closely related specificities, i.e. subfamilies [11, 30], as well as many examples of close evolutionary relatedness were reported for the individual groups of α-amylases, e.g., those from animals and actinomycetes [24], plants and archaeons [31, 32], insects [33], and fungi [34, 35].

Exogenous and endogenous inhibitory proteins have also been reported from microorganisms and plants [36] directed towards α-amylases [37] and limit dextrinases [38, 39, 40].

Kinetics and Mechanism

GH13 enzymes employ the retaining mechanism. This was first demonstrated by quantitative gas liquid chromatographic analysis of formation of α-maltose from different maltosides [41] and further supported by the NMR analysis of the release of α-maltose from similar substrates [42]. It was also demonstrated for a number of different α-amylases that they follow the classical Koshland double-displacement mechanism. This has moreover been supported by covalent labelling using 4-deoxy-maltotriose-fluoride trapping the catalytic nucleophile [43], numerous three-dimensional structures, and site-directed mutational substitution of the catalytic site residues [44, 45]. Some of the GH13 members use a multiple attack or processive mechanism [46, 47, 48] involving several glycoside bond cleavages to be executed in the same enzyme-substrate encounter. In several cases the binding energies have been determined by using subsite mapping [49, 50, 51, 52], which gives a subsite binding energy profile characteristic for individual enzymes. Several α-amylases have been reported to interact with polymeric substrates at surface sites situated as a certain distance of the active site [53, 54, 55, 56]. Finally interaction with insoluble substrates, such as starch granules or glycogen can occur both at these sites [54, 55, 57] as well as by the involvement of separate binding modules referred to as starch binding domains [58, 59, 60, 61].

Catalytic Residues

The catalytic residues have been identified from early crystal structures [62, 63]. In fact throughout the family GH13 only the catalytic triad plus an arginine residue are totally conserved; the catalytic site includes an aspartate as catalytic nucleophile, a glutamate as general acid/base, and an aspartate that participates critically in stabilizing the transition state [43]. The fourth invariantly conserved GH13 residue, the arginine, is positioned two residues preceding the catalytic nucleophile [4]. This conservation does not apply for the enzymatically inactive heavy-chains (rBAT proteins and 4F2hc antigens) of the amino acid transporters [8]. Numerous mutational analyses have been performed to confirm the essential roles of the 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. In general, the GH13 members are multidomain proteins with catalytic (β/α)8-barrel (i.e. TIM-barrel) domain (called domain A) having a small domain B (usually varying in length and of irregular structure) [6] inserted in the loop between the β3-strand and α3-helix of the barrel, and succeeded by the C-terminal antiparallel β-sandwich domain, called domain C. The catalytic site formed by the C-terminal extensions of strands β4, β5 and β7, carrying the catalytic triad of aspartate, glutamate and aspartate, respectively [62, 64, 65], but also other loops contribute to the overall architecture of the active site.

The first crystals for barley α-amylase were reported in the mid-forties, however the first crystal structures were those of TAKA-amylase A [62, 66] and porcine pancreatic α-amylase [63, 64]. This was followed by structures of other α-amylases from bacteria [67, 68, 69] and from higher plants [65, 70]; the industrially important cyclodextrin glucanotransferase [71, 72, 73] and the closely related maltogenic α-amylase [74]. Later on the structures of the amylopectin debranching isoamylase [75] and the related pullulanase [76] and limit dextrinase [77] were determined. Furthermore the oligo-1,6-glucosidase [78] and the related dextran glucosidase [79], as well as maltogenic amylase [80], cyclomaltodextrinase [81] and neopullulanase [82] - nearly indistinguishable from each other - together with the neopullulanase-like “α-amylases” TVA I [83] and TVA II [84], and the amylosucrase [85], sucrose phosphorylase [86], sucrose hydrolase [87] and sucrose isomerase [88, 89], were solved. Finally structures have been solved of glycogen branching [90] and debranching [91] enzymes.

Among the solved structures are numerous site-directed-mutant and ligand-complexed forms. However, although there are structures available for most of the GH13 specificities, some still remain to be determined. Noticeably crystal structures are available of several α-amylase/proteinaceous inhibitor complexes (for reviews, see [37, 92]).

Family Firsts

First sterochemistry determination

α-Maltose was released from different α-maltosides by Bacillus subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, as determined by quantitative gas liquid chromatography [41]. This was as well demonstrated by NMR analysis of the anomeric configuration of the released product [42].

First catalytic nucleophile

A glycosidic covalent bond was formed in the intermediate of the mechanism between the catalytic nucleophile (D229) of Bacillus circulans 251 CGTase and a maltotriosyl moiety [43]. Mutational analysis of human pancreatic α-amylase provided strong support for D197 being the catalytic nucleophile as demonstrated by kinetics analysis [93].

First general acid/base

Mutational 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 [93].

First 3-D structure

The first high-resolution three-dimensional structure was determined for Taka-amylase A [62].

References

All Medline abstracts: PubMed

@@ Line 56: / Line 56: @@
 == Family Firsts ==
 ;First sterochemistry determination
-α-Maltose was released from different α-maltosides by ''Bacillus subtilis'' saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, as determined by quantitative gas liquid chromatography <cite>Kimura1983</cite>. This was as well demonstrated by NMR analysis of the anomeric configuration of the released product <cite>Isoda1982</cite>.
+α-Maltose was released from different α-maltosides by ''Bacillus subtilis'' saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, as determined by quantitative gas liquid chromatography <cite>Kimura1983</cite>. This was as well demonstrated by NMR analysis of the anomeric configuration of the released product <cite>Isoda1992</cite>.
 ;First [[catalytic nucleophile]]
 A glycosidic covalent bond was formed in the intermediate of the mechanism between the catalytic nucleophile (D229) of ''Bacillus circulans'' 251 CGTase and a maltotriosyl moiety <cite>Uitdehaag1999</cite>. Mutational analysis of human pancreatic α-amylase provided strong support for D197 being the catalytic nucleophile as demonstrated by kinetics analysis <cite>Rydberg2002</cite>.