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

Abstract

Glycoside hydrolases (GHs) have been classified in the CAZy database into 153 GH families. Currently, there might be four α-amylase families: the main family GH13, the family GH57 with related GH119 and, eventually, also GH126. The family GH57 was established in 1996 as the second and smaller α-amylase family. In addition to α-amylase, it contains 4-α-glucanotransferase, α-glucan branching enzyme, amylopullulanase, dual-specificity amylopullulanase–cyclomaltodextrinase, non-specified amylase, maltogenic amylase and α-galactosidase. The family GH57 enzymes employ the retaining reaction mechanism, share five typical conserved sequence regions and possess catalytic (β/α)7-barrel succeeded by a four-helix bundle with the catalytic machinery consisting of catalytic nucleophile and proton donor (glutamic acid and aspartic acid at strands β4 and β7, respectively). The present bioinformatics study delivers a detailed sequence comparison of 1602 family GH57 sequences with the aim to highlight the uniqueness of each enzyme’s specificity and all eventual protein groups. This was achieved by creating the evolutionary tree focused on both the enzyme specificities and taxonomical origin. The substantial increase of numbers of sequences from recent comparisons done more than 5 years ago has allowed to refine the details of the sequence logos for the individual enzyme specificities. The study identifies a new evolutionary distinct group of α-galactosidase-related enzymes with until-now-undefined enzyme specificity but positioned on the evolutionary tree on a branch adjacent to α-galactosidases. The specificity of α-galactosidase is, moreover, the only one of the entire family GH57 for which there is no structural support for the proposal of the proton donor based on sequence analysis. The analysis also suggests a few so-called “like” protein groups related to some family GH57 enzyme specificities but lacking one or both catalytic residues.Electronic supplementary materialThe online version of this article (10.1007/s13205-018-1325-9) contains supplementary material, which is available to authorized users.

Identification of Thermotoga maritima MSB8 GH57 α-amylase AmyC as a glycogen-branching enzyme with high hydrolytic activity

“…Analysis of the GH57 GBE sequences and phylogenetic tree construction was performed as described in the ‘Materials and methods’ section. Sequences of 40 GBEs (Supplementary Table S1) were collected based on the recent exhaustive in silico analysis of the entire α-amylase family GH57 (Martinovičová and Janeček 2018) that, of all 1602 GH57 sequences taken from the CAZy database (Cantarel et al 2009), yielded 546 GBEs. Forty GBEs were selected in an effort to obtain a representative sample of GBE sequences having, in addition to AmyC from T. maritima (Ballschmiter et al 2006; Dickmanns et al 2006), the biochemically characterized enzymes from T. kodakaraensis (Murakami et al 2006; Santos et al 2011), T. thermophilus (Palomo et al 2011) and P. horikoshi (Na et al 2017), accompanied by a range of hypothetical GBEs covering various taxa from both Bacteria and Archaea including all available sequences from the phylum Thermotogae .…”

“…Forty GBEs were selected in an effort to obtain a representative sample of GBE sequences having, in addition to AmyC from T. maritima (Ballschmiter et al 2006; Dickmanns et al 2006), the biochemically characterized enzymes from T. kodakaraensis (Murakami et al 2006; Santos et al 2011), T. thermophilus (Palomo et al 2011) and P. horikoshi (Na et al 2017), accompanied by a range of hypothetical GBEs covering various taxa from both Bacteria and Archaea including all available sequences from the phylum Thermotogae . In order to perform as relevant as possible analysis and in accordance with previous in silico studies (Blesak and Janecek 2012; Blesak and Janecek 2013; Janecek and Blesak 2011; Martinovičová and Janeček 2018), the set of sequences was completed by 23 biochemically characterized family GH57 members representing other enzyme specificities, accompanied by one putative representative of the α-amylase-like protein (Supplementary Table S1).…”

“…For all 64 GH57 enzymes and proteins (Supplementary Table S1), their five well-established conserved sequence regions (CSRs) (Zona et al 2004) were identified according to previous bioinformatics analyses (Blesak and Janecek 2012, 2013; Janecek and Blesak 2011; Martinovičová and Janeček 2018). The evolutionary tree was calculated based on the alignment of five CSRs mentioned above as a Phylip-tree type using the neighbour-joining clustering (Saitou and Nei 1987) and the bootstrapping procedure (Felsenstein 1985) (the number of bootstrap trials used was 1000) implemented in the Clustal-X package (Larkin et al 2007).…”

“…For the family GH57 members, five conserved sequence regions (CSRs) have been established (Zona et al 2004). Currently, GH57 holds over 2000 protein sequences (CAZy update from February 2019) comprising hydrolytic and transglycosylating enzymes, such as α-amylase, amylopullulanase (EC 3.2.1.41), dual-specificity amylopullulanase/cyclomaltodextrinase (EC 3.2.1.41/54), glycogen-branching enzyme (GBE; EC 2.4.1.18), 4-α-glucanotransferase, and α-galactosidase (EC 3.2.1.22), as well as a non-specified amylase (EC 3.2.1.-) and maltogenic amylase (EC 3.2.1.133) (Blesak and Janecek 2012; Blesak and Janecek 2013; Janecek et al 2014; Martinovičová and Janeček 2018; Zona et al 2004). Glycogen-branching enzymes of GH57 play a pivotal role in the synthesis of glycogen, cleaving an α-1,4-glycosidic linkage in the donor substrate subsequently transferring the non-reducing end fragment to the C6 hydroxyl position of an internal glucosyl moiety that acts as the acceptor substrate (α-1,6-transglycosylation).…”

Extension of the taxonomic coverage of the family GH126 outside Firmicutes and in silico characterization of its non-catalytic terminal domains

“…In the sequence-based classification of glycoside hydrolases (GHs) of the CAZy database ( https://www.cazy.org/ ; Lombard et al 2014 ), the family GH126 was established after the study by Ficko-Blean et al ( 2011 ) delivering the three-dimensional structure and partial characterization as a potential α-amylase of the protein CPF_2247 from Clostridium perfringens ATCC 13124 genome (Myers et al 2006 ). The structure was solved as that of a typical catalytic (α/α) 6 -barrel fold known, e.g., in the family GH15 glucoamylases (Sauer et al 2000 ; Kumar and Satyanarayana 2009 ; Marin-Navarro and Polaina 2011 ), but adopted neither by α-amylases from families GH13 and GH57 and even in GH119 (Janecek and Kuchtova 2012 ; Janecek et al 2014 ; Martinovicova and Janecek 2018 ), nor by β-amylases from family GH14 (Monroe and Storm 2018 ).…”