Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57

Abstract

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.

In silico analysis of the α-amylase family GH57: eventual subfamilies reflecting enzyme specificities

“…1 a). The tree contains clusters of family GH57 enzymes, such as α-amylase, 4-α-glucanotransferase, amylopullulanase, bifunctional amylopullulanase–cyclomaltodextrinase, maltogenic amylase, α-galactosidase, non-specified amylase and α-glucan branching enzyme, well established by previous studies (Blesak and Janecek 2012 , 2013 ). With regard to their mutual evolutionary relationships, α-amylase are clustered together with 4-α-glucanotransferase; both being in a closer relatedness with amylopullulanases and their bifunctional counterparts possessing also the cyclomaltodextrinase specificity.…”

“…Of these, 1568 sequences were retrieved from the family GH57 of the CAZy database directly, whereas remaining 34 sequences of the specificity of maltogenic amylase were obtained using the BLAST. This was because the three biochemically characterized maltogenic amylases have still not been classified within the family GH57, although previous in silico analysis (Blesak and Janecek 2013 ) along with cloning, sequencing and structural studies (Comfort et al 2008 ; Jeon et al 2014 ; Jung et al 2014 ; Park et al 2014 ) have clearly suggested they exhibit all sequence/structural features characteristic of the family GH57.…”

“…Sequences were collected according to the information for the family GH57 in the CAZy database (Lombard et al 2014 ), except for the specificity of maltogenic amylase (or maltose-forming amylases) that as yet has not been assigned to any CAZy family despite the fact that it was demonstrated to exhibit all the sequence-structural features characteristic of the family GH57 (Blesak and Janecek 2013 ; Jeon et al 2014 ; Jung et al 2014 ; Park et al 2014 ). The sequences of maltogenic amylases, currently kept in CAZy among the “non-classified” sequences, were obtained by protein BLAST search (Altschul et al 1990 ) using the sequence of maltogenic amylase from Pyrococcus sp.…”

Diversity of Glycosyl Hydrolase Enzymes from Metagenome and Their Application in Food Industry

“…Wang and others (), Ballschmiter and others (), and Zhang and Zeng () reported on amylases from many different extreme environments. In an effort to understand the mechanism of action of GH 57 amylase family, in relation to structure, Blesak and Janecek () studied 2 partially characterized GH 57 nonspecified amylases from an uncultured bacterium by bioinformatics analysis. Though the study found the enzyme hydrolyzing soluble starch, specificities and the reaction products could not be determined.…”

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

“…Twenty years ago, several α-amylases and related enzymes composed of a (β/α) 7 -barrel (an irregular TIM-barrel domain) were classified into the family GH57; more recently also the family GH119 was established910. Both of these enzyme families are at present considerably smaller than GH13 and only few members have been characterized in detail11.…”