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Glycoside Hydrolase Family 57

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Glycoside Hydrolase Family GH57
Clan not assigned yet
Mechanism retaining
Active site residues known/not known
CAZy DB link

Substrate specificities

The family GH57 was established in 1996 [1] based on the existence of the sequences of two “α-amylases” that were dissimilar to typical family GH13 α-amylases [2]. The two were the heat-stable eubacterial amylase from Dictyoglomus thermophilum known from 1988 [3] and the extremely thermostable archaeal amylase from Pyrococcus furiosus determined in 1993 [4].

The family has expanded mainly due to running genome sequencing projects. Nowadays it contains more than 400 members; all originating from prokaryotes ( With regard to the enzyme specificities, the family GH57 covers the α-amylase (EC, α-galactosidase (EC, amylopullulanase (EC, branching enzyme (EC and 4-α-glucanotransferase (EC It is worth mentioning that the two constituent members, i.e. the “α-amylases” from D. thermophilum and P. furiosus are rather the 4-α-glucanotransferases since the former was later proven to have the transglycosylating activity [5], whereas the latter was shown already in 1993 to exhibit the 4-α-glucanotransferase activity [6]. And it is also of interest that the real enzymes form only about 5% of the family members. The vast majority of the GH57 are hypothetical proteins.

Kinetics and Mechanism

Family GH57 are retaining enzymes, as first documented by the X-ray crystallography on the 4-α-glucanotransferase from Thermococcus litoralis complexed with acarbose [7]. Kinetic studies have been performed with the 4-α-glucanotransferases from Thermococcus litoralis [7, 8], Pyrococcus furiosus [9], amylopullulanases from Thermococcus hydrothermalis [10] and Pyrococcus furiosus [11] and branching enzyme from Thermococcus kodakaraensis [12].

Catalytic Residues

In addition, the sequences of GH57 members are extremely diversified. Certain sequences are shorter than 400 residues whereas others are longer than 1,500 residues [13]. This complicated the previous efforts to align the GH57 sequences using the routine alignment programs. Based on a detailed bioinformatics study focused on all available GH57 sequences at that time, five conserved sequence regions in the family GH57 were identified and proposed by [10]. This was possible to achieve since the catalytic nucleophile (Glu123) in the GH57 4-α-glucanotransferase from Thermococcus litoralis [8] was known together with its three-dimensional structure [7] (PDB: 1k1w) that revealed also the proton donor (Asp214).

The catalytic nucleophile (a glutamate) and proton donor (an aspartate) are located in the conserved sequence regions 3 and 4, respectively. In addition to Thermococcus litoralis 4-α-glucanotransferase, they were identified also in the amylopullulanases from Thermococcus hydrothermalis [10] and Pyrococcus furiosus [11]. The catalytic nucleophile was confirmed also in the α-galactosidase from Pyrococcus furiosus although without success to find the catalytic proton donor [14]. It should be taken into account, however, that some GH57 members, which are only hypothetical enzymes/proteins without any biochemical characterization, may lack one or even both catalytic residues [10].

Based on the five identified conserved sequence regions, the residues His13, Glu79, Glu216, Asp354 together with the Trp120, Trp221 and Trp357 (Thermococcus hydrothermalis 4-α-glucanotransferase numbering) were postulated [10] as eventually important for the individual GH57 enzyme specificities. Of these, the Trp221 has already been confirmed to contribute to the transglycosylation activity of 4-α-glucanotransferase since the mutant W229H of the enzyme from Pyrococcus furiosus exhibited markedly decreased transglycosylation activity in comparison with the wild-type counterpart [9].

Three-dimensional structures

The structure of the catalytic domain adopts a (β/α)7-barrel, i.e. the irregular (β/α)8-barrel called also a pseudo TIM-barrel that, in the case of the Thermococcus litoralis 4-α-glucanotransferase [7] is succeeded by the C-terminal non-catalytic domain consisting of β-strands only adopting a twisted β-sandwich fold. In the three-dimensional structure of the α-amylase AmyC from Thermotoga maritima [15] (PDB: 2b5d), the corresponding catalytic (β/α)7-barrel is followed by a five-helix domain C, a small helical domain B being protruded out of the catalytic pseudo TIM barrel in the place of the loop 2 (i.e. succeeding the strand β2). This structure was found to be most closely similar to that of the GH57 member of unknown function from Thermus thermophilus (PDB: 1ufa). In all cases, the catalytic glutamic acid and aspartic acid residues are located near the C-terminal ends of the strands β4 and β7 of the barrel, respectively [7, 15]. There was also a crystallization report in 1995 on a probable GH57 amylopullulanase from Pyrococcus woesei [16], but the detailed crystallographic analysis of this protein has not been published as yet.

It is clear that the C-terminal domain cannot be present in some GH57 members with shorter amino acid sequences, e.g., in the α-galactosidases containing less than 400 residues [14]. On the other hand, some other GH57 members, especially the extra-long amylopullulanases with more than 1,300 residues [17] have to contain even additional domains. One of them is a longer version of a typical SLH motif (surface layer homology) [18] that was named as the so-called SLH motif-bearing domain in the amylopullulanase from Thermococcus hydrothermalis [17]. This domain was found also in the GH15 glucodextranase from Arthrobacter globiformis [19]. Remarkably, within the family GH57, the presence of this SLH motif-bearing domain is restricted only for amylopullulanases [20].

It is also worth mentioning that, especially prior the first three-dimensional structure of a GH57 member was available, there were some efforts to join the family GH57 with the main α-amylase family GH13, i.e. the present clan GH-H consisting of the families GH13, GH70 and GH77 [2]. Those efforts were focused mainly on looking for some remote homology at the sequence level only [21, 22]. Although both GH57 and GH-H employ the same retaining reaction mechanism [7, 23] the independence of the family GH57 with regard to GH-H clan is at present based not only on differences in the catalytic domain, but more importantly, due to differences in the catalytic machineries and conserved sequence regions [10, 24]. As far as other GH families are concerned, the family GH38 α-mannosidase from Drosophila melanogaster [25] was revealed to share some structural similarities within the catalytic domain with the GH57 4-α-glucanotransferase from Thermococcus litoralis [7, 8] indicating an eventuality of originating from a common ancestor.

Family Firsts

First sterochemistry determination
Probably the work on the 4-α-glucanotransferase from Thermococcus litoralis [7] or that on branching enzyme from Thermococcus kodakaraensis [12].
First amino acid sequence determination
The first amino acid sequence of the family GH57 was that of a heat stable amylase from an anaerobic thermophilic bacterium Dictyoglomus thermophilum [3]. This "α-amylase" was later characterized as 4-α-glucanotransferase [5].
First conserved sequence regions determination
The five sequence stretches characteristic as conserved regions for the family GH57 were first determined by the bioinformatics study by [10].
First catalytic nucleophile identification
The catalytic nucleophile was fist identified by [8] as Glu123 in the 4-α-glucanotransferase from Thermococcus litoralis using the 3-ketobutylidene-β-2-chloro-4-nitrophenyl maltopentaoside as a donor.
First general acid/base residue identification
Asp214 of the 4-α-glucanotransferase from Thermococcus litoralis as indicated by the X-ray crystallography and supported by site-directed mutagenesis [7] since the D214N mutant exhibited a 10,000-fold decrease of specific activity in comparison with the wild-type enzyme).
First 3-D structure
The first 3-D structure of a GH57 member was that of the 4-α-glucanotransferase from Thermococcus litoralis [7].


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All Medline abstracts: PubMed | HubMed