A novel gellan gum-degrading glycoside hydrolase from the hyperthermophile Fervidibacter sacchari

Fervidibacter sacchari is an aerobic hyperthermophile within the phylum Armatimonadota that degrades various polysaccharides. Its genome encodes 117 putative glycoside hydrolases (GHs), including two assigned to GH family 50 (GH50). In this study, a multidomain GH50 enzyme, Fsa11540Gus, was expressed, purified, and functionally characterized. Despite most GH50s being known as β-agarases, Fsa11540Gus was optimally active on gellan gum, followed by β-glucan, with lower activity on carboxymethyl (CM)-curdlan and agarose. Nanostructure-initiator mass spectrometry (NIMS) analysis of degradation products of gellan gum indicated β-1,4-endoglucuronidase activity at the glucuronate-glucose bond. Mutation of either conserved active-site glutamic acid with proposed roles as acid/base catalyst and nucleophile to alanine (E409A or E575A) or amino acids with predicted roles in substrate recognition and distortion (N408A, F630A, and E646A) decreased enzyme activity. These results rule out gellan lyase activity and confirm Fsa11540Gus as the first known glycoside hydrolase to degrade gellan gum. NIMS analysis of other degradation products revealed β-1,3-endoglucanase activity on oat β-glucan and CM-curdlan and likely β-1,4-endo glycoside hydrolase activity on the galactose-3,6-anhydrogalactose-galactose bond in agarose. The enzyme was active on gellan gum from 70 to 95 °C, with optimal activity at 80–90 °C and rapid deactivation at 100 °C. AlphaFold 3 computed structure models predicted a monomeric structure, which was confirmed by native polyacrylamide gel electrophoresis. The enzyme has a predicted N-terminal Sec translocase/signal peptidase I signal peptide, an N-terminal domain of unknown function, a GH50 domain, and a large C-terminal domain annotated as COG1572/pfam07705. Phylogenetic analysis of Fsa11540Gus and other GH50 enzymes revealed a monophyletic clade in the genera Fervidibacter and Caldisaccharidevorator with these distinctive N- and C-terminal domains, suggesting recent vertical evolution but a dynamic deeper evolutionary history involving domain swapping and changes in protein localization. This study shows that Fsa11540Gus is the first known enzyme with glycoside hydrolase activity on gellan gum and expands our understanding of the GH50 family and polysaccharide metabolism by Armatimonadota .