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

The family GH126 is a family of glycoside hydrolases established in 2011. Officially, in the CAZy database, it counts ~ 1000 sequences originating solely from bacterial phylum Firmicutes. Two members, the proteins CPF_2247 from Clostridium perfringens and PssZ from Listeria monocytogenes have been characterized as a probable α-amylase and an exopolysaccharide-specific glycosidase, respectively; their three-dimensional structures being also solved as possessing catalytic (α/α)6-barrel fold. Previously, based on a detailed in silico analysis, the seven conserved sequence regions (CSRs) were identified for the family along with elucidating basic evolutionary relationships within the family members. The present study represents a continuation study focusing on two particular aims: (1) to find out whether the taxonomic coverage of the family GH126 might be extended outside the Firmicutes and, if positive, to deliver those out-of-Firmicutes proteins with putting them into the context of the family; and (2) to identify the family members containing the N- and/or C-terminal extensions of their polypeptide chain, additional to the catalytic (α/α)6-barrel domain, and perform the bioinformatics characterization of the extra domains. The main results could be summarized as follows: (1) 17 bacterial proteins caught by BLAST searches outside Firmicutes (especially from phyla Proteobacteria, Actinobacteria and Bacteroidetes) have been found and convincingly suggested as new family GH126 members; and (2) a thioredoxin-like fold and various leucine-rich repeat motifs identified by Phyre2 structure homology modelling have been recognized as extra domains occurring most frequently in the N-terminal extensions of family GH126 members possessing a modular organization.

A detailed in silico analysis of the amylolytic family GH126 and its possible relatedness to family GH76.

The glycoside hydrolase (GH) family 126 was established based on the X-ray structure determination of the amylolytic enzyme CPF_2247 from Clostridium perfringens genome. Its original identification as a putative carbohydrate-active enzyme was based on its low, yet significant sequence identity to members of the family GH8, which are inverting endo-ß-1,4-glucanases. As the family GH8 forms the clan GH-M with GH48, the CPF_2247 protein also exhibits similarities with members of the family GH48. The original screening of the CPF_2247 on carbohydrate substrates demonstrated its activity on glycogen and amylose, thus classifying this protein as an "α-amylase". It should be pointed out, however, there are apparent inconsistencies concerning the exact enzyme specificity of the "amylase" CPF_2247, since it exhibits both the endo- and exo-fashion of action. The family GH126 currently counts ~1000 amino acid sequences solely from Bacteria; all belonging to the phylum Firmicutes. The present study delivers the first detailed bioinformatics study of 117 selected amino acid sequences from the family GH126, featuring the insightful sequence-structure comparison with the aim to define seven conserved sequence regions and elucidate the evolutionary relationships within the family. In addition, a comparative structural analysis of the GH126 members with representatives of other GH families adopting the same (α/α)6-barrel catalytic domain fold indicates the possible sharing a catalytic residue between the families GH126 and GH76.