Glycoside Hydrolase Family 136

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

• Author: Chihaya Yamada
• Responsible Curator: Shinya Fushinobu

---

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 liberates 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 liberates Galβ1-3GalNAc (GNB) from the sugar chains of globo- and ganglio-series glycosphingolipids [2].

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

Kinetics and Mechanism

GH136 lacto-N-biosidases hydrolyze the glycosidic linkage via anomer-retaining mechanism. The 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 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 nucleophile and the catalytic 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

1. Sakurama H, Kiyohara M, Wada J, Honda Y, Yamaguchi M, Fukiya S, Yokota A, Ashida H, Kumagai H, Kitaoka M, Yamamoto K, and Katayama T. (2013). 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. J Biol Chem. 2013;288(35):25194-25206. DOI:10.1074/jbc.M113.484733 |
2. Gotoh A, Katoh T, Sugiyama Y, Kurihara S, Honda Y, Sakurama H, Kambe T, Ashida H, Kitaoka M, Yamamoto K, and Katayama T. (2015). Novel substrate specificities of two lacto-N-biosidases towards β-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1. Carbohydr Res. 2015;408:18-24. DOI:10.1016/j.carres.2015.03.005 |
3. Pichler MJ, Yamada C, Shuoker B, Alvarez-Silva C, Gotoh A, Leth ML, Schoof E, Katoh T, Sakanaka M, Katayama T, Jin C, Karlsson NG, Arumugam M, Fushinobu S, and Abou Hachem M. (2020). Butyrate producing colonic Clostridiales metabolise human milk oligosaccharides and cross feed on mucin via conserved pathways. Nat Commun. 2020;11(1):3285. DOI:10.1038/s41467-020-17075-x |
4. Yamada C, Gotoh A, Sakanaka M, Hattie M, Stubbs KA, Katayama-Ikegami A, Hirose J, Kurihara S, Arakawa T, Kitaoka M, Okuda S, Katayama T, and Fushinobu S. (2017). Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum. Cell Chem Biol. 2017;24(4):515-524.e5. DOI:10.1016/j.chembiol.2017.03.012 |
5. Yamada C, Katayama T, and Fushinobu S. (2022). Crystal structures of glycoside hydrolase family 136 lacto-N-biosidases from monkey gut- and human adult gut bacteria. Biosci Biotechnol Biochem. 2022;86(4):464-475. DOI:10.1093/bbb/zbac015 |