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
http://www.cazy.org/GH47.html

Substrate specificities

GH47 glycoside hydrolases are exo-acting α-1,2-mannosidases. Members from this family play important roles in the processing of N-glycans and are classified as Class I mannosidases in contrast to the Class II mannosidases of GH38 [1]. Three subfamilies of GH47 enzymes have been identified based upon their different substrate specifities.

In mammalian species, ER-α-mannosidase I is representative of the GH47 subfamily that acts upon Man₉GlcNAc₂ to cleave M10 and afford the Man₈GlcNAc₂ isomer B. Extended incubation results in further demannosylated products in vitro [2]. Pulse-chase studies have found that Saccharomyces cerevisiae α-mannosidase I, the only GH47 mannosidase of the organism, bears the same activity as mammalian ER-α-mannosidase I, encouraging the use of this enzyme as a model for the mammalian enzyme[1].

In mammalian species, the GH47 Golgi mannosidase I subfamily acts on Man_8-9GlcNAc₂ to afford Man₅GlcNAc₂ isomer C and is composed of 3 members (denoted IA, IB and IC) [3]. In contrast to mammalian ER-α-mannosidase I, the Golgi-resident GH47 mannosidases preferentially cleave from the A and C chains of the glycan. Depending on the Golgi mannosidase I subfamily member, M9 or M11 is removed first, followed by M8 [4]. All mammalian Golgi mannosidase I enzymes tested thus far have low relatively activity against M10, meaning that GH47 mannosidases from the ER and Golgi have complementary actitivities.

Fewer studies have focussed upon the role of GH47 enzymes in plants. However, it has been found that these mannosidases are essential for N-glycan processing and are important for normal root developmemt and cell wall biosynthesis in Arabidopsis thaliana [5].

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Kinetics and Mechanism

GH47 mannosidases catalyze glycosidic cleavage with inversion of stereochemistry, as first determined employing ¹H NMR spectroscopy with Saccharomyces cervisiae α-1,2-mannosidase using Man9GlcNAc as a substrate [6]. Classical inverting glycosidases operate through a single displacement mechanism, where a general base residue acts to deprotonate a water molecule, facilitating nucleophilic attack at the anomeric position. This is assisted by concurrent activation of the glycosidic linkage through protonation by a general acid residue.

GH47 enzymes are Ca²⁺-dependent, as demonstrated by loss of activity upon addition of the metal binding ligand EDTA, and restoration of activity through subsequent addition of Ca²⁺ [7]. Exo-α-mannosidases from GH38 and GH92 also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary. Structural studies employing unhydrolysable S-linked substrate analogues have examined the Michaelis complex, with the ligands found to bind in ³S₁ [8] and ^3,OB/³S₁ conformations [9]. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry [10], binds GH47 in a ³H₄ conformation [9]. Noeuromycin [9], kifunensine [11] and 1-deoxymannojirimycin [11] all bind in a ¹C₄ conformation, analogous to enzyme-product complexes. Computational studies also support a ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary [9, 12, 13]. Quantum mechanical/molecular modelling calculations have found that the free energy landscape of α-D-mannopyranose is perturbed on-enzyme such that the accessible conformations of the ligand are altered to those that correlate well with a ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary [9].

Catalytic Residues

Unequivocal assignment of catalytic residues for GH47 α-mannosidases is complicated by the presence of 3 carboxylate-containing residues all approximately 9.5 Å apart from one another in the active site. Each of these could plausibly fulfill roles as catalytic residues [14]. 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. Site directed mutagenesis of residues in the α-mannosidase I of Aspergillus saitoi and Saccharomyces cerevisiae predated determination of a crystal structure but demonstrated that mutation of any of the three catalytic candidates led to total or near-total loss of activity [15, 16]. Mutagenesis of residues in human ER α-mannosidase I, informed by the determination of the crystal structure, could not unambiguously assign the role of catalytic residues [8]. Glu132 (Glu330 in human ER α-mannosidase I) in Saccharomyces cerevisiae α-mannosidase I was initially thought to be most likely candidate as the general base residue [14]. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual ¹C₄ conformation [11]. These complexes were interpreted as being representative of a ¹C₄ Michaelis complex, making Glu330 (Glu132 in Saccharomyces) incompatible with a role acting as the general base in an inverting mechanism. Thus, the general base residue was reassigned as either Glu599 or Asp463 (Glu435 and Asp275 in Saccharomyces, respectively). A computational docking study found Glu599 to be the most likely general base, with Ca²⁺ also coordinated to the nucelophilic water molecule [17]. However, complexes with S-linked substrate analogues implicate a ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary, the reverse of that used to preclude Glu330 (Glu132 in Saccharomyces) as the general base residue [8, 9]. The position of Glu330 (Glu132 in Saccharomyces) on the opposite face of the glycan ring to the putative general base residue, Glu599 in human ER α-mannosidase I (Glu435 in Saccharomyces), is consistent with a role as the general acid [11]. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad [8]. 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 [18]. The low nanomolar binding of mannoimidazole to CkGH47 is consistent with anti-protonation [9].

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 [14]. The –1 subsite lies in the core of the barrel with Ca²⁺ coordinating to the 2-OH and 3-OH groups of a ligand (inhibitor or substrate analogue), whose glycan ring is parallel to the barrel upon complexation [11].

The structural basis for differences in N-glycan branch specificity between ER and Golgi GH47 α-mannosidases has been examined through crystallographic studies comparing their binding to N-glycans [19]. 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 [6].
First general base identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [14]. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in S. cerevisiae) [17].
First general acid identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [14]. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in S. cerevisiae) [11], however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in S. cerevisiae) [18].
First 3-D structure: Saccharomyces cerevisiae α-1,2-mannosidase [14].

References

All Medline abstracts: PubMed