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Glycoside Hydrolase Family 57
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|Glycoside Hydrolase Family GH57|
|Clan||not assigned yet|
|Active site residues||known/not known|
|CAZy DB link|
The family GH57 was established in 1996 (Henrissat & Bairoch 1996) based on the existence of the sequences of two “α-amylases” that were dissimilar to typical family GH13 α-amylases (MacGregor et al. 2001). The two were the heat-stable eubacterial amylase from Dictyoglomus thermophilum known from 1988 (Fukusumi et al. 1988) and the extremely thermostable archaeal amylase from Pyrococcus furiosus determined in 1993 (Laderman et al. 1993a).
The family has expanded mainly due to running genome sequencing projects. Nowadays it contains more than 400 members; all originating from prokaryotes (http://www.cazy.org/fam/GH57.html). With regard to the enzyme specificities, the family GH57 covers the α-amylase (EC 18.104.22.168), α-galactosidase (EC 22.214.171.124), amylopullulanase (EC 126.96.36.199/41), branching enzyme (EC 188.8.131.52) and 4-α-glucanotransferase (EC 184.108.40.206). 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 (Nakajima et al. 2004), whereas the latter was shown already in 1993 to exhibit the 4-α-glucanotransferase activity (Laderman et al., 1993b). 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 . Kinetic studies have been performed with the 4-α-glucanotransferases from Thermococcus litoralis [1, 2], Pyrococcus furiosus , amylopullulanases from Thermococcus hydrothermalis  and Pyrococcus furiosus  and branching enzyme from Thermococcus kodakaraensis .
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 (Janecek 2005). 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 (Fig. 1) were identified and proposed by Zona et al. (2004). This was possible to achieve since the catalytic nucleophile (Glu123) in the GH57 4-α-glucanotransferase from Thermococcus litoralis (Imamura et al. 2001) was known together with its three-dimensional structure (Imamura et al. 2003; PDB: 1k1w) that revealed also the proton donor (Asp214). Thus the first catalytic machinery and the first three-dimensional structure for a GH57 member (Fig. 2) were those of the archaeal 4-α-glucanotransferase from T. litoralis (Imamura et al. 2001, 2003).
The catalytic nucleophile (a glutamate) and proton donor (an aspartate) are located in the conserved sequence regions 3 and 4, respectively (Fig. 1). In addition to T. litoralis 4-α-glucanotransferase, they were identified also in the amylopullulanases from Thermococcus hydrothermalis (Zona et al. 2004) and P. furiosus (Kang et al. 2005). The catalytic nucleophile was confirmed also in the α-galactosidase from P. furiosus although without success to find the catalytic proton donor (van Lieshout et al. 2003). 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 (Zona et al. 2004).
Based on the five identified conserved sequence regions (Fig. 1), the residues His13, Glu79, Glu216, Asp354 together with the Trp120, Trp221 and Trp357 (T. hydrothermalis 4-α-glucanotransferase numbering) were postulated (Zona et al. 2004) as eventually important for the individual GH57 enzyme specificities (Fig. 3). 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 P. furiosus exhibited markedly decreased transglycosylation activity in comparison with the wild-type counterpart (Tang et al. 2006).
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 (Fig. 2) that, in the case of the T. litoralis 4-α-glucanotransferase (Imamura et al. 2003) is succeeded by the C-terminal non-catalytic domain consisting of β-strands only adopting a twisted β-sandwich fold (Fig. 2). In the three-dimensional structure of the α-amylase AmyC from Thermotoga maritima (Dickmanns et al. 2006; PDB: 2b5d), the corresponding catalytic (β/α)7-barrel is followed by a five-helix domain C and a small helical domain B protruding 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 (Imamura et al. 2003, Dickmanns et al. 2006). There was also a crystallization report in 1995 on a probable GH57 amylopullulanase from Pyrococcus woesei (Knapp et al. 1995), 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 (van Lieshout et al. 2003). On the other hand, some other GH57 members, especially the extra-long amylopullulanases with more than 1,300 residues (Erra-Pujada et al. 1999) have to contain even additional domains. One of them is a longer version of a typical SLH motif (surface layer homology) (Lupas et al. 1994) that was named as the so-called SLH motif-bearing domain in the amylopullulanase from T. hydrothermalis (Erra-Pujada et al. 1999). This domain was found also in the GH15 glucodextranase from Arthrobacter globiformis (Mizuno et al. 2004). Remarkably, within the family GH57, the presence of this SLH motif-bearing domain is restricted only for amylopullulanases (Zona & Janecek 2005).
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 (MacGregor et al. 2001). Those efforts were focused mainly on looking for some remote homology at the sequence level only (Dong et al. 1997; Janecek 1998). Although both GH57 and GH-H employ the same retaining reaction mechanism (Imamura et al 2003; Matsuura et al. 1984) 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 (Zona et al. 2004; Janecek 2002). As far as other GH families are concerned, the family GH38 α-mannosidase from Drosophila melanogaster (van den Elsen et al. 2001) was revealed to share some structural similarities within the catalytic domain with the GH57 4-α-glucanotransferase from T. litoralis (Imamura et al. 2001, 2003) indicating an eventuality of originating from a common ancestor.
- First sterochemistry determination
- 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 . This "α-amylase" was later characterized as 4-α-glucanotransferase .
- 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 .
- First catalytic nucleophile identification
- The catalytic nucleophile was fist identified by  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  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 .