Glycoside Hydrolase Family 127

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• Author: Kiyotaka Fujita
• Responsible Curator: Shinya Fushinobu

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Glycoside Hydrolase Family GH127
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH127.html

Substrate specificities

This family of glycoside hydrolases contains β-L-arabinofuranosidase activity, which was established for HypBA1 from Bifidobacterium longum JCM 1217 [1]. HypBA1 released L-arabinose from the following saccharides and amino acid glycoconjugates, but not from from hydroxyproline-rich glycoproteins (HRGPs) such as carrot extensin and potato lectin:

• Arafβ1-2Araf (β-Ara₂, a product of the GH121 β-L-arabinobiosidase from B. longum JCM 1217 [2])
• Arafβ-hydroxyproline (Ara-Hyp)
• Arafβ1-2Arafβ-Hyp (Ara₂-Hyp)
• Arafβ1-2Arafβ1-2Arafβ-hyp (Ara₃-Hyp)
• methyl β-L-arabinofuranoside
• Arafβ1-2Arafβ-Me

The members of GH127 were previosuly known as members of the Pfam DUF1680 family, which is conserved in many species of bacteria, actinomycetes, fungi, and plants. Establishment of GH127 by biochemical analysis thus resolved the "domain of unknown function" status of this PFAM family.

Note: The original paper describing B. longum HypBA1 as the founding member of GH127 [3] was withdrawn in 2013 [4] and a revised version was published in 2014 [1], to correct errors due to an inadvertent mix-up of sample tubes and data for the E338A and E366A mutants.

Kinetics and Mechanism

HypBA1 is a retaining enzyme. The stereochemical course of the reaction was shown by transglycosylation activity toward 1-alkanols, such as methanol, and produced methyl β-L-arabinofuranoside was identified by ¹H-NMR and ¹³C-NMR analysis [1]

Catalytic Residues

In the crystal structure of HypBA1, a Zn²⁺ ion was bound to the active site [5]. A cysteine residue (Cys417), which is involved in the coordination of the Zn²⁺, was suggested to act as the nucleophile. Glu322 is possibly the acid/base catalyst. A possible reaction mechanism involving the cysteine residue as the nucleophile was suggested based on crystal structures, site-directed mutagenesis, some biochemical analysis, and quantum mechanical calculations [5].

Three-dimensional structures

HypBA1 from B. longum JCM 1217 [5]. It consists of a catalytic (α/α)₆ barrel domain and two additional β-sandwich domains.

Family Firsts

First stereochemistry determination

This was determined with HypBA1 enzyme by measurement of glycosyl transfer reactions to methanol and the ¹H-NMR and¹³C-NMR spectra [3].

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

HypBA1 from B. longum JCM 1217 by X-ray crystallography [5].

References

1. Fujita K, Takashi Y, Obuchi E, Kitahara K, and Suganuma T. (2014). Characterization of a novel β-L-arabinofuranosidase in Bifidobacterium longum: functional elucidation of a DUF1680 protein family member. J Biol Chem. 2014;289(8):5240-9. DOI:10.1074/jbc.M113.528711 |
2. Fujita K, Sakamoto S, Ono Y, Wakao M, Suda Y, Kitahara K, and Suganuma T. (2011). Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family. J Biol Chem. 2011;286(7):5143-50. DOI:10.1074/jbc.M110.190512 |
3. Fujita K, Takashi Y, Obuchi E, Kitahara K, and Suganuma T. (2011). Characterization of a novel β-L-Arabinofuranosidase in Bifidobacterium longum: functional elucidation of A DUF1680 family member. J Biol Chem. 2011;286(44):38079-38085. DOI:10.1074/jbc.M111.248690 |
4. Fujita K, Takashi Y, Obuchi E, Kitahara K, and Suganuma T. (2013). Characterization of a novel β-L-arabinofuranosidase in Bifidobacterium longum. Functional elucidation of a DUF1680 family member. J Biol Chem. 2013;288(42):30502. DOI:10.1074/jbc.A111.248690 |
5. Ito T, Saikawa K, Kim S, Fujita K, Ishiwata A, Kaeothip S, Arakawa T, Wakagi T, Beckham GT, Ito Y, and Fushinobu S. (2014). Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum. Biochem Biophys Res Commun. 2014;447(1):32-7. DOI:10.1016/j.bbrc.2014.03.096 |