Glycoside Hydrolase Family 57
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|Glycoside Hydrolase Family GH57|
|Clan||not assigned yet|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolase family 57 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 the results of genome sequencing projects. More than a thousand of members are known [5, 6], all from prokaryotes (with ~1:4 ratio for Archaea:Bacteria) . The enzyme specificities of family GH57 includes α-amylase (EC 184.108.40.206), α-galactosidase (EC 220.127.116.11), amylopullulanase (EC 18.104.22.168), branching enzyme (EC 22.214.171.124) and 4-α-glucanotransferase (EC 126.96.36.199).
An archaeal GH57 amylopullulanase from Staphylothermus marinus has been described exhibiting also the activity of cyclodextrinase (EC 188.8.131.52) . Based on a preliminary observation that the PF0870 protein encoded in the Pyrococcus furiosus genome produced maltose , a group of GH57 members with proposed specificity of maltogenic amylase (EC 184.108.40.206) was predicted  together with another group of non-specified amylases following the partial characterization of an amylolytic enzyme from an uncultured bacterium . The maltogenic specificity (or maltose-forming amylase) has already been definitively confirmed by both biochemical [12, 13] and structural  studies. The family GH57 contains further a group of probably non-enzymatic members, i.e. the so-called α-amylase-like proteins having a substitution in one or both catalytic residues .
It is worth mentioning that the two founding members, i.e. the “α-amylases” from Dictyoglomus thermophilum and Pyrococcus furiosus are 4-α-glucanotransferases; the former was proven to have transglycosylating activity , whereas the latter was already known to exhibit also the 4-α-glucanotransferase activity .
A detailed in silico study has indicated a close relatedness of family GH57 to the family GH119 concerning catalytic domain fold, catalytic machinery and conserved sequence regions .
Kinetics and Mechanism
A direct evidence for the stereochemical outcome of the enzyme-catalyzed reaction has been presented for the branching enzyme from Thermus thermophilus  by the NMR analysis of reaction products. The previous observation of a trapped covalent glycosyl-enzyme intermediate  was also a strong evidence that the GH57 enzymes operate with retention of anomeric configuration through a classical Koshland retaining mechanism with retention of anomeric configuration. The X-ray structure of the 4-α-glucanotransferase from Thermococcus litoralis revealed average distances of 6.72 Å and 6.97 Å between the catalytic nucleophile (Glu123) and general acid/base (Asp214) in the free and acarbose-bound forms of the enzyme, respectively, which also supports a retaining mechanism for this family  (see also ).
Detailed kinetic studies have been performed on several GH57 enzymes, including the 4-α-glucanotransferases from Thermococcus litoralis [20, 21] and Pyrococcus furiosus , amylopullulanases from Thermococcus hydrothermalis  and Pyrococcus furiosus , and branching enzymes from Thermococcus kodakaraensis  and Thermus thermophilus .
The sequences of GH57 members are very diverse. Some sequences are shorter than 400 residues whereas others are longer than 1,500 residues . This complicated early efforts to align the GH57 sequences using standard alignment methods. A detailed bioinformatics study by Zona et al.  focused on all available GH57 sequences at that time and identified five conserved sequence regions. This study used knowledge of the identity of the catalytic nucleophile (Glu123) in the GH57 4-α-glucanotransferase from Thermococcus litoralis  together with the three-dimensional structure  (PDB ID 1k1w) that revealed the general acid/base residue (Asp214).
The catalytic nucleophile (a glutamate) and general acid/base (an aspartate) are located in the conserved sequence regions 3 and 4, respectively. In addition to assignments in Thermococcus litoralis 4-α-glucanotransferase discussed above, these residues were identified in the amylopullulanases from Thermococcus hydrothermalis  and Pyrococcus furiosus . The catalytic nucleophile was also identified in the α-galactosidase from Pyrococcus furiosus although no success was achieved in assigning the general acid/base . It should be taken into account that some GH57 members, which are only hypothetical enzymes/proteins without any biochemical characterization, may lack one or even both catalytic residues [15, 24].
Based on the five identified conserved sequence regions, the residues His13, Glu79, Glu216 and Asp354 together with Trp120, Trp221 and Trp357 (Thermococcus hydrothermalis amylopullulanase numbering) were postulated  as important determinants of the individual GH57 enzyme specificities [7, 10, 15]. 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 a pseudo TIM-barrel. In the case of the Thermococcus litoralis 4-α-glucanotransferase  the catalytic domain is succeeded by a C-terminal non-catalytic domain consisting of only β-strands that adopts a twisted β-sandwich fold. In the three-dimensional structure of the α-amylase AmyC from Thermotoga maritima  (PDB ID 2b5d) (note that the sequence of this enzyme strongly resemles that of a branching enzyme ), 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 closely similar to that of the GH57 member of unknown function from Thermus thermophilus (PDB ID 1ufa). Two structures of biochemically characterized branching enzymes were solved and published: one from Thermus thermophilus  (PDB ID 3p0b) and the other one from Thermococcus kodakaraensis  (PDB ID 3n8t). The former study , importantly, is the first report to prove that a retaining mechanism is employed in the family GH57. Also, the 3-D structure is available for the maltogenic (maltose-forming) amylase from Pyrococcus sp. ST04  (PDB ID 4cmr). 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 [21, 29]. 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.
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 could be a longer version of a typical surface layer homology (SLH) motif  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 [36, 37]. Although both GH57 and GH-H employ the same retaining reaction mechanism [21, 38] 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, on the differences in the catalytic machineries and conserved sequence regions [24, 39]. 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 [20, 21] indicating an eventuality of originating from a common ancestor.
- First sterochemistry determination
- The first direct evidence of the retaining reaction stereochemistry by product analysis has been delivered by 1H-NMR analysis of the product mixture that confirmed the α-anomeric configuration of the α-1,6-glucosidic bond formed after the incubation of Thermus thermophilus branching enzyme with amylose . Previously, family GH57 enzymes have been indicated to be retaining by trapping of a covalent glycosyl-enzyme intermediate  and a 6.7 Å distance between the catalytic nucleophile and acid/base , both of which are consistent with a two-step, double-displacement mechanism.
- First amino acid sequence determination
- The first amino acid sequence of the family GH57 was that of the heat stable amylase from an anaerobic thermophilic bacterium Dictyoglomus thermophilum . This "α-amylase" was later characterized as 4-α-glucanotransferase . In fact, the family contains many α-amylase-like proteins, i.e. those that exhibit a clear sequence homology with α-amylases, but possess a substitution in one or both catalytic residues .
- 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 Zona et al. (2004) .
- First catalytic nucleophile identification
- The catalytic nucleophile was fist identified by Imamura et al. (2001)  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 .
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