Shizuoka University

Flounder Roe Triggering α-Gal Syndrome: Hyosophorin as a Possible Allergen.

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

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.

Regioselective synthesis of p-nitrophenyl glycosides of beta-D-galactopyranosyl-disaccharides by transglycosylation with beta-D-galactosidases.

The beta-D-galactosidase from porcine liver induced regiospecific transglycosylation of beta-D-galactose from beta-D-Gal-OC6H4NO2-o to OH-6 of, respectively, p-nitrophenyl glycoside acceptors of Gal, GlcNAc and GalNAc to afford beta-Gal-(1-->6)-alpha-Gal-OC6H4NO2-p, beta-Gal-(1--> 6)-beta-Gal-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-beta-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GlcNAc-OC6H4NO2-p, and beta-Gal-(1-->6)-beta-GlcNAc-OC6H4NO2-p. The enzyme showed much higher transglycosylation activity for the alpha-glycoside acceptors than the corresponding beta-glycoside acceptors. The regioselectivity of the beta-D-galactosidase from Bacillus circulans ATCC 31382 greatly depended on the nature of the acceptor. When alpha-D-GalNAc-OC6H4NO2-p and alpha-D-GlcNAc-OC6H4NO2-p were used as acceptors, the enzyme showed high potency for regioselective synthesis of beta-Gal-(1-->3)-alpha-GalNAc-OC6H4NO2-p and beta-Gal-(1-->3)-alpha-GlcNAc-OC6H4NO2-p in high respective yields of 75.9 and 79.3% based on the acceptors added. However, replacement of beta-D-Gal-OC6H4NO2-p by beta-D-GalNAc-OC6H4NO2-p did change the direction of galactosylation. The enzyme formed regioselectively beta-Gal-(1-->6)-beta-Gal-OC6H4NO2-p with (beta-Gal-1-->(6-beta-Gal-1-->)n6-beta-Gal-OC6H4NO2-p, n = 1-4). No beta-(1-->3)-linked product was detected during the reaction. Use of the two readily available beta-D-galactosidases facilitates the preparation of (1-->3)- and (1-->6)-linked disaccharide glycosides of beta-D-Gal-GalNAc and beta-D-Gal-GlcNAc.