Glycoside Hydrolase Family 47

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Author: ^^^Rohan Williams^^^
Responsible Curator: ^^^Spencer Williams^^^

Glycoside Hydrolase Family GHnn
Clan	none, (α/α)₇ fold
Mechanism	inverting
Active site residues	debated
CAZy DB link
https://www.cazy.org/GH47.html

Substrate specificities

GH47 enzymes can be divided into 3 subfamilies based upon their role in the maturation of N-glycans.

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Kinetics and Mechanism

GH47 mannosidases catalyse glycosidic cleavage with inversion of stereochemistry, as first determined through 1H NMR studies using Saccharomyces cervisiae α-1,2-mannosidase with Man9GlcNAc as a substrate [1]. GH47 enzymes are Ca²⁺-dependent,at present only fellow exo-α-mannosidases from GH38 and GH92 are known to also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary, supported by both computational [2, 3] and structural studies [3, 4, 5]. Quantum mechanical/molecular modelling calculations have found that the free energy landscape of α-D-mannose is perturbed on-enzyme such that the accessible conformations of the ligand are restricted to those that correlate well with a ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary [3].

Catalytic Residues

Unequivocal assignment of catalytic residues for GH47 α-mannosidases is complicated by the presence of 3 carboxylate-containing residues in the active site who could all plausibly fulfill roles as catalytic residues [6]. Furthermore, all of the plausible catalytic residues complex water, as would be expected of the general base residue. Thus, it appears that the general acid residue transmits a proton to the glycosidic oxygen atom through a water molecule. Crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxynojirimycin found that an inverting mechanism was only compatible with Glu599 or Asp463 (Glu435 and Asp275 in Saccharomyces, respectively) acting as the general base [4]. A computational docking study found Glu599 to be the most likely general base, with Ca²⁺ also coordinated to the nucelophilic water molecule [7]. Based upon its position on the opposite face of the glycan ring to the potential general base residues in human ER a-mannosidase I, Glu330 (Glu132 in Saccharomyces) is widely believed to act as the general acid [4]. However, a computational docking study found Asp463 (Asp275 in Saccharomyces) to be the most likely general acid, based upon the assumption that GH47 mannosidases are anti-protonators [8].

Three-dimensional structures

GH47 enzymes adopt a (α/α)₇ barrel fold with a Ca²⁺ ion coordinated at the base of the barrel that is plugged by a β-hairpin at the C-terminus [6]. The –1 subsite lies in the core of the barrel with Ca²⁺ coordinating to the 2-OH and 3-OH groups of a ligand, whose glycan ring is parallel to the barrel upon complexation[4].

The structural basis for differences in branch specificity between ER and Golgi GH47 α-mannosidases has been examined through crystallographic studies comparing their binding to N-glycans [9]. The presumed enzyme-product complexes differed in their oligosaccharide conformation such that different oligosaccharide branches, corresponding to those readily cleaved by the respective enzymes, were projected into the active site.

Family Firsts

First sterochemistry determination: Saccharomyces cerevisiae α-1,2-mannosidase was shown to be inverting by ¹H NMR [1].
First general base identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [6]. Widely believed to be Glu559 in human ER α-mannosidase I (Glu435 in S. cerevisiae) [7].
First general acid identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [6]. Widely believed to be Glu330 in human ER α-mannosidase I (Glu132 in S. cerevisiae) [5], however, a computational study concluded that it was Asp463 in human ER α-mannosidase I (Asp275 in S. cerevisiae) [8].
First 3-D structure: Saccharomyces cerevisiae α-1,2-mannosidase [6].

References

All Medline abstracts: PubMed