The glycoside hydrolase family 57 (GH57) contains five well-established enzyme specificities: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase. Around 700 GH57 members originate from Bacteria and Archaea, a substantial number being produced by thermophiles. An intriguing feature of family GH57 is that only slightly more than 2 % of its members (i.e., less than 20 enzymes) have already been biochemically characterized. The main goal of the present bioinformatics study was to retrieve from databases, and analyze in detail, sequences having clear features of the five GH57 enzyme specificities mentioned above. Of the 367 GH57 sequences, 56 were evaluated as α-amylases, 99 as amylopullulanases, 158 as branching enzymes, 46 as 4-α-glucanotransferases and 8 as α-galactosidases. Based on the analysis of collected sequences, sequence logos were created for each specificity and unique sequence features were identified within the logos. These features were proposed to define the so-called sequence fingerprints of GH57 enzyme specificities. Domain arrangements characteristic of the individual enzyme specificities as well as evolutionary relationships within the family GH57 are also discussed. The results of this study could find use in rational protein design of family GH57 amylolytic enzymes and also in the possibility of assigning a GH57 specificity to a hypothetical GH57 member prior to its biochemical characterization.

The glycoside hydrolase family 57 (GH57) contains α-amylase and a few other amylolytic specificities. It counts ~400 members from Archaea (1/4) and Bacteria (3/4), mostly of extremophilic prokaryotes. Only 17 GH57 enzymes have been biochemically characterized. The main goal of the present bioinformatics study was to analyze sequences having the clear GH57 α-amylase features. Of the 107 GH57 sequences, 59 were evaluated as α-amylases (containing both GH57 catalytic residues), whereas 48 were assigned as GH57 α-amylase-like proteins (having a substitution in one or both catalytic residues). Forty-eight of 59 α-amylases were from Archaea, but 42 of 48 α-amylase-like proteins were of bacterial origin. The catalytic residues were substituted in most cases in Bacteroides and Prevotella by serine (instead of catalytic nucleophile glutamate) and glutamate (instead of proton donor aspartate). The GH57 α-amylase specificity has thus been evolved and kept enzymatically active mainly in Archaea.

Glycoside hydrolase (GH) family 57 consists of more than 900 proteins from Archaea (roughly one-quarter) and Bacteria (roughly three-quarters), mostly from thermophiles. Fewer than 20 GH57 members have already been biochemically characterized as real, (almost exclusively) amylolytic enzymes. In addition to a recently described dual-specificity amylopullulanasecyclomaltodextrinase, five enzyme specificities have been well established in the familya-amylase, amylopullulanase, branching enzyme, 4-a-glucanotransferase and a-galactosidaseplus a group of the so-called a-amylase-like homologues probably without the enzyme activity. A (b/a) 7 -barrel succeeded by a bundle of a few a-helices forming the catalytic domain, and five conserved sequence regions (CSRs), are the main characteristics of family GH57. The main goal of the present bioinformatics study was to describe two novel groups within family GH57 that represent potential non-specified amylases (127 sequences mostly from Bacteria) and maltogenic amylases (12 sequences from Archaea). These were collected from sequence databases based on an indication of their biochemical characterization. Although both the nonspecified amylases and the maltogenic amylases share the in silico identified catalytic machinery and predicted fold with the experimentally determined GH57 members, the two novel groups may define new GH57 subfamilies. They are distinguishable from the other, previously recognized, subfamilies by specific sequence features present especially in their CSRs (the so-called sequence fingerprints), also reflecting their own evolutionary histories.