Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

Abstract

Background: Infant gut-associated bifidobacteria possess lacto-N-biosidase, which releases lacto-N-biose I (LNB) from human milk oligosaccharides. Results: The crystal structures of lacto-N-biosidase complexed with LNB and LNB-thiazoline were determined. Conclusion: The intermediate analog complex allows the proposal of a conformational reaction coordinate. Significance: The structures of a key enzyme in the colonization of human commensal bacteria provided its structural basis and insight into the development of inhibitors.

Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

“…Interestingly, we found that this strain possesses no gene homologous to the previously identified LNBase (glycoside hydrolase family 20, GH20) from B. bifidum (17). Although the B. longum JCM1217 genome encodes a GH20 enzyme, the protein obviously lacks the sequence motifs to distinguish LNBase from ␤-N-acetylhexosaminidase (another GH20 member) (28). Actually, the GH20 enzyme of B. longum JCM1217 was recently found to be ␤-N-acetylglucosaminidase active on lacto-N-triose II (GlcNAc␤1-3Gal␤1-4Glc) and on chitin oligosaccharides but not on LNT (29).…”

“…4). The activity of GH20 LNBase from B. bifidum (LnbB) was also evaluated for comparison (17,28). LnbX hydrolyzed both LNB-␤-pNP and GalNAc␤1-3GlcNAc␤-pNP, whereas LnbB was inactive toward GalNAc␤1-3GlcNAc␤-pNP (Table 3).…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…Based on amino acid homology, ␤-N-acetylhexosaminidases are classified into glycoside hydrolase (GH) families 3, 20, 84, and 123 (15)(16)(17)(18)(19)(20)(21). Among them, GH3 enzymes generally employ a double-displacement mechanism, in which a pair of acidic residues serve as catalytic acid/base and nucleophile residues (15,16).…”

“…Among them, GH3 enzymes generally employ a double-displacement mechanism, in which a pair of acidic residues serve as catalytic acid/base and nucleophile residues (15,16). In contrast, GH20, GH84, and GH123 enzymes employ the substrate-assisted mechanism in which the 2-acetamido group of the substrate acts as a nucleophile to form an oxazoline intermediate for catalysis (17)(18)(19)(20)(21). A large number of bifunctional ␤-N-acetylhexosaminidases from the GH20 family show hydrolysis activity toward both GalNAc-and GlcNAc-containing substrates (17,18), whereas GH3, GH84, and GH123 enzymes specifically hydrolyze GalNAc-or GlcNAc-containing substrates (15,16,(19)(20)(21).…”

“…GH20 enzymes contain a pair of highly conserved catalytic residues, namely, AspGlu; Glu acts as an acid/base residue, whereas Asp serves as a nucleophile assistant residue that assists the substrate 2-acetamido group to act as a nucleophile (15). In the case of BbhI, the residues Asp714 and Glu715 were predicted as nucleophile assistant and catalytic acid/base amino acids, whereas the residues His647, Trp743, Trp769, and Trp850 can be considered essential for enzyme activity based on comparison with the corresponding residues of other reported ␤-N-acetylhexosaminidases (18,46). The putative 3D model of BbhI suggests that these predicted residues are responsible for enzyme activity (Fig.…”