2.5.2. Auxiliary Activity Family 3 Enzymes

According to the CAZyme annotation (Figure 5 and Table S1), AA3 is characterized by a multiplicity of members (up to five candidate proteins) for the *Zobellia* genus. AA3 belongs to the glucose-methanol-choline (GMC) oxidoreductase family first outlined by Cavener [61]. It was reported that GMC oxidoreductases were flavoproteins containing FAD-binding domain with the strictly conserved Rossmann fold or β-α-β dinucleotide-binding motif GXGXXG [62]. Our results demonstrate that proteins in the studied *Zobellia* strains predicted as AA3 enzymes also have such motif, suggesting that they may act as GMC oxidoreductases (Figure S3).

The GMC oxidoreductases are a very large and functionally diverse enzyme superfamily divided into four subfamilies, which include cellobiose dehydrogenases, glucose oxidoreductases, aryl-alcohol oxidases, alcohol oxidases, and pyranose oxidoreductases [63]. In 2012, Kim et al. [64] identified GMC oxidoreductase from a *Rhizobium* sp. strain GIN611 with glycoside deglycosylation activity different from that of common glycosidases (GHs). Later [65] they characterized its homologs in *Stenotrophomonas maltophilia*, *Sphingobacterium multivorum*, and *Agrobacterium tumefaciens* strains, catalyzed the deglycosylation via the same mechanism, and suggested these enzymes as a new GMC oxidoreductase subfamily—FAD-dependent glycoside oxidoreductase (FAD-GO). Interestingly, the authors showed broad glycone and aglycon specificities for these enzymes that makes them very attractive in their industrial applications.

We performed a phylogenetic analysis by comparing the amino acid sequences of *Zobellia* AA3 members with characterized FAD-GOs. According to the phylogenetic tree (Figure S4), the *Zobellia* enzymes formed two clades, one of which (Clade A) was orthologous to the FAD-GO proteins. Sequence comparison of *Zobellia* Clade A enzymes and glycoside oxidoreductases revealed relatively high identity between them (53%–58%) (Table S2). Moreover, His493 residue considered as a catalytic FAD-GO amino acid for an initial oxidation step was observed within protein sequences for all studied *Zobellia* (Figure S3). This gives us the opportunity to suggest that *Zobellia* AA3 enzymes could have the same glycoside oxidase activity, identification of which may be the subject for further research. Due to the broad substrate specificity, putative *Zobellia* FAD-GOs are of particular interest and can be considered as promising biocatalysts for glycoside deglycosylation in food and pharmaceutical industries [65].
