Structure–function analysis of bacterial GH31 α‐galactosidases specific for α‐(1→4)‐galactobiose

Abstract

Glycoside hydrolase family 31 (GH31) contains α‐glycoside hydrolases with different substrate specificities involved in various physiological functions. This family has recently been classified into 20 subfamilies using sequence similarity networks. An α‐galactosidase from the gut bacterium Bacteroides salyersiae (BsGH31_19, which belongs to GH31 subfamily 19) was reported to have hydrolytic activity against the synthetic substrate p‐ nitrophenyl α‐galactopyranoside, but its natural substrate remained unknown. BsGH31_19 shares low sequence identity (around 20%) with other reported GH31 α‐galactosidases, PsGal31A from Pseudopedobacter saltans and human myogenesis‐regulating glycosidase (MYORG), and was expected to have distinct specificity. Here, we characterized BsGH31_19 and its ortholog from a soil Bacteroidota bacterium, Flavihumibacter petaseus (FpGH31_19), and demonstrated that they showed high substrate specificity against α‐(1→4)‐linkages in α‐(1→4)‐galactobiose and globotriose [α‐Gal‐(1→4)‐β‐Gal‐(1→4)‐Glc], unlike PsGal31A and MYORG. The crystallographic analyses of BsGH31_19 and FpGH31_19 showed that their overall structures resemble those of MYORG and form a dimer with an interface different from that of PsGal31A and MYORG dimers. The structures of FpGH31_19 complexed with d‐galactose and α‐(1→4)‐galactobiose revealed that amino acid residues that recognize a galactose residue at subsite +1 are not conserved between FpGH31_19 and BsGH31_19. The tryptophan (Trp153) that recognizes galactose at subsite −1 is homologous to the tryptophan residues in MYORG and α‐galactosidases belonging to GH27, GH36, and GH97, but not in the bacterial GH31 member PsGal31A. Our results provide structural insights into molecular diversity and evolutionary relationships in the GH31 α‐galactosidase subfamilies and the other α‐galactosidase families.