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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  based on the existence of the sequences of two “α-amylases” that were dissimilar to typical family GH13 α-amylases . The two were the heat-stable eubacterial amylase from Dictyoglomus thermophilum known from 1988  and the extremely thermostable archaeal amylase from Pyrococcus furiosus determined in 1993 .
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 184.108.40.206), α-galactosidase (EC 220.127.116.11), amylopullulanase (EC 18.104.22.168/41), branching enzyme (EC 22.214.171.124) and 4-α-glucanotransferase (EC 126.96.36.199). 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 , whereas the latter was shown already in 1993 to exhibit the 4-α-glucanotransferase activity . 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 [7, 8], 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 . 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 . This was possible to achieve since the catalytic nucleophile (Glu123) in the GH57 4-α-glucanotransferase from Thermococcus litoralis  was known together with its three-dimensional structure  (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  and Pyrococcus furiosus . The catalytic nucleophile was confirmed also in the α-galactosidase from Pyrococcus furiosus although without success to find the catalytic proton donor . 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 .
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  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 .
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  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  (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 , 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 . On the other hand, some other GH57 members, especially the extra-long amylopullulanases with more than 1,300 residues  have to contain even additional domains. One of them is a longer version of a typical SLH motif (surface layer homology)  that was named as the so-called SLH motif-bearing domain in the amylopullulanase from Thermococcus hydrothermalis . This domain was found also in the GH15 glucodextranase from Arthrobacter globiformis . Remarkably, within the family GH57, the presence of this SLH motif-bearing domain is restricted only for amylopullulanases .
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 . 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  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.
- First sterochemistry determination
- Probably the work on the 4-α-glucanotransferase from Thermococcus litoralis  or that on branching enzyme from Thermococcus kodakaraensis .
- 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 .
- Imamura H, Fushinobu S, Yamamoto M, Kumasaka T, Jeon BS, Wakagi T, and Matsuzawa H. (2003). Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor. J Biol Chem. 2003;278(21):19378-86. DOI:10.1074/jbc.M213134200 |
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Fukusumi, S., Kamizono, A., Horinouchi, S. & Beppu, T. 1988. Eur. J. Biochem. 174: 15-21.
Laderman, K.A., Asada, K., Uemori, T., Mukai, H., Taguchi, Y., Kato, I. & Anfinsen, C.B. 1993a. J. Biol. Chem. 268: 24402-24407.
Nakajima, M., Imamura, H., Shoun, H., Horinouchi, S. & Wakagi, T. 2004. Biosci. Biotechnol. Biochem. 68: 2369-2373.
Laderman, K.A., Davis, B.R., Krutzsch, H.C., Lewis, M.S., Griko. Y.V., Privalov, P.L. & Anfinsen, C.B. 1993b. J. Biol. Chem. 268: 24394-24401.