Glycoside Hydrolase Family 136

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• Author: Chihaya Yamada
• Responsible Curator: Shinya Fushinobu

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Glycoside Hydrolase Family GH136
Clan	GH-N
Mechanism	retaining
Active site residues	Asp
CAZy DB link
https://www.cazy.org/GH136.html

Substrate specificities

This family of glycoside hydrolases contains lacto-N-biosidase, as demonstrated for LnbX from Bifidobacterium longum JCM 1217 [1]. LnbX liberated Galβ1-3GlcNAc (lacto-N-biose I, LNB) and lactose from lacto-N-tetraose, the main component of human milk oligosaccharides. It hydrolyzed the linkage GlcNAcβ1-3Gal in lacto-N-hexaose, lacto-N-fucopentaose I, and sialyllacto-N-tetraose a of human milk oligosaccharides as substrate of LnbX in the GH136. In addition, LnbX liberated Galβ1-3GalNAc (GNB) from the sugar chains of globo- and ganglio-series glycosphingolipids [2].

The majority of GH136 lacto-N-biosidases require a neighboring chaperone gene for folding. Rarely, the chaperone-like gene is fused to the lacto-N-biosidase gene, as in case of ErLnb136_I and ErLnb136_II from Eubacterium ramulus [3].

Kinetics and Mechanism

GH136 lacto-N-biosidases hydrolyze the glycosidic linkage via a anomer-retaining mechanism. The general acid/base catalytic residue of LnbX (Asp411) formed a water-mediated hydrogen bond with the O1 atom of GlcNAc at subsite -1, and a mechanism of Grotthuss proton transfer was proposed [4]. However, subsequent crystallographic reports on three GH136 lacto-N-biosidases ("Er"Lnb136, BsaX, and TnX) revealed a direct hydrogen bond between the general acid/base catalyst and the O1 atom. This observation suggests that a direct proton transfer mechanism is prevalent within this family [3, 5].

Catalytic Residues

For LnbX, the catalytic nucleophile and the catalytic general acid/base are Asp418 and Asp411, respectively.

Three-dimensional structures

Figure 1: Overall structure of LnbXc with LNB (cyan) and two Ca2+ ions (orange).

Figure 2: Overall structure of ErLnb136 with LNB (yellow), consisting of an N-terminal domain designated as ErLnb136_I (cyan-blue) and a C-terminal β-helix domain (green) -ErLnb136_II.

The X-ray crystal structure of the catalytic domain, LnbXc(31-625) revealed a right-handed β helix fold that is usually shared by polysaccharide-active enzymes. Three forms, ligand free at 2.36 Å resolution (PDB ID 5GQC), LNB complex at 1.82 Å (PDB ID 5GQF), and GNB complex at 2.70 Å (PDB ID 5GQG) were determined [4]. The X-ray crystal structure of ErGH136 in complex with LNB (PDB ID 6KQT) revealed the N-terminal domain (ErLnb136I, from AA 7-224) consists of 8 α-helices (α1-α8) and Y145 of the α6-α7 loop positioned near the active site [3]. The LNB-complexed structures of the catalytic domain of BsaX from Bifidobacterium saguini and TnX from Tyzzerella nexilis were also reported [5].

Family Firsts

First stereochemistry determination

LnbX from Bifidobacterium longum [1].

First catalytic nucleophile identification

LnbX from Bifidobacterium longum [4].

First general acid/base residue identification

LnbX from Bifidobacterium longum [4].

First 3-D structure

LnbX from Bifidobacterium longum [4].

References

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