Glycoside hydrolases (GHs) are carbohydrate-active enzymes catalyzing the hydrolysis of glycosidic bonds in saccharides employing either retaining or inverting mechanism [1]. The family GH126 was established based on the X-ray structure determination of the amylolytic enzyme CPF_2247 from Clostridium perfringens ATCC 13124 genome [2]. Its original identification was based on its low, yet significant sequence identity (~20%) to members of the family GH8, which are inverting endo-β-1,4-glucanases [3]. As the family GH8 forms the clan GH-M with GH48 [4], the CPF_2247 protein also exhibits similarities with members of the family GH48 [2]. The screening of the CPF_2247 on carbohydrate substrates demonstrated interestingly its activity on glycogen and amylose, thus classifying this protein as an “α-amylase” [2]. It should be pointed out, however, there are apparent inconsistencies concerning the exact enzyme specificity of the “α-amylase” CPF_2247, since it exhibits both the endo- and exo-mode of action [2]. Moreover, there is a difficulty of understanding how a retaining α-amylase could share the catalytic mechanism with above-mentioned inverting β-glucanases [5]. The family GH126 currently counts more than 750 members, whereas the Clostridium perfringens “α-amylase” is still the only characterized enzyme in the family GH126. It is thus of interest to shed some light on sequence-structural details and evolutionary relatedness of this potentially novel α-amylase family, especially with regard to the existence of the main α-amylase family GH13 (the clan GH-H) [5,6] and the two additional α-amylase families GH57 and GH119 [7,8].

The present in silico study tries to shed some light on the less characterized family GH126 and its possible evolutionary connections within the CAZy database. The sequence alignment allows for identification of possible CSRs; these could be beneficial in distinguishing future sequences and their categorization as a GH126 family member. The sequence alignment also led to the creation of an evolutionary tree with branch organization that suggest further categorization of GH126 sequences into 9 groups; further research is needed to distinguish possible protein function differences of these sequences. A major part of the study has been focused on a comparative structural analysis of the “α-amylase” CPF_2247 with representatives of other GH families in the CAZy database [1] that share the same catalytic (α/α)₆-barrel fold in an effort to reveal potential evolutionary relatedness.The structural analysis proved its structural relatedness with β-glucanases of families GH8 and GH48 as well as suggest a evolutionary connection with members of the retaining α-mannanase family GH76.