The family GH126 – its relatedness to and differentiation from GH8 and GH48 including the intermediary sequences

Abstract

The family GH126, best represented by the amylolytic enzyme CPF_2247 from Clostridium perfringens, exclusively includes proteins of bacterial origin, covering predominantly the phylum Bacillota. Although all the members should adopt the catalytic (α/α)6-barrel domain, neither the catalytic machinery nor the reaction mechanism has been determined as yet. The limited biochemical characterization, especially some uncertainty concerning the endo- vs exo-mode of action and retaining vs inverting mechanism, combined with the sequence-structural resemblance of GH126 members to inverting β-glucanases from families GH8 and GH48 (the clan GH-M), may lead to misclassification of putative proteins. The present study was therefore designed in an effort to identify unique sequence-structural features that would definitively differentiate family GH126 from both GH8 and GH48. To achieve this, a sequence logo, representing the seven GH126 conserved sequence regions established previously, was created using 1,665 GH126 sequences. The logo was compared with GH8 and GH48 logos based on, respectively, 86 and 63 selected enzymes. An invariant tyrosine residue in CSR-6 was identified as a reliable marker for the family GH126. In addition, protein BLAST searches identified 87 putative proteins that taxonomically extend the family GH126 not only outside Bacillota, but also outside Bacteria to include representatives of archaeons and eukaryotes (fungi). Evolutionary analysis of the 434 sequences representing all the three families GH126 with GH8 and GH48, including the BLAST hits, revealed an intermediate group. In the future, it may either define a new GH family closely related to GH126, or at least constitute a future GH126 subfamily.