Glycoside Hydrolase Family 47

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

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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 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 (ERMI) is representative of the GH47 subfamily that acts upon Man₉GlcNAc₂ to cleave a mannose from the B-chain affording Man₈GlcNAc₂. 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 essentially the same activity as mammalian ER-α-mannosidase I [1].

In mammalian species, the GH47 Golgi mannosidase I (Golgi MI) subfamily acts on Man_8-9GlcNAc₂ to afford Man₅GlcNAc₂ 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 in an order that depends on the subfamily member [4]. All mammalian Golgi mannosidase I enzymes tested thus far have relatively low activity against the B-chain of the glycan, meaning that GH47 mannosidases from the ER and Golgi have complementary actitivities.

The third GH47 subfamily is composed of the ER degradation-enhancing mannosidase-like (EDEM) proteins. This subfamily is composed of 3 members in humans and was initially not thought to have direct glycosidase activity. However, it now appears that the EDEM1 and EDEM3 isoforms have glycosidase activity in vivo [5, 6]. All of these isoforms have been shown to accelerate the disposal of terminally misfolded proteins through ER-associated degradation (ERAD) [6, 7, 8]. The process of recognition of terminally misfolded proteins and the role of EDEM proteins is not fully understood, however, it is believed that EDEM proteins function as lectins, binding after demannosylation of the glycan. A current model for entry of glycoproteins into ERAD states that correct folding mediated by the calnexin folding cycle must occur before the slow demannosylation of the substrate affords Man₆GlcNAc₂, which is no longer a substrate for reglucosylation by UGGT1 and re-entry into the calnexin folding cycle [9]. It is not clear which mannosidases perform this extensive demannosylation in vivo. Upon extensive demannosylation to Man_5-6GlcNAc₂ structures it is proposed that EDEM proteins facilitate retro-translocation of the glycoprotein to the cytosol and its subsequent proteolysis, although the exact role of EDEM proteins has yet to be elucidated.

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 in Arabidopsis thaliana [10].

Schematic depicting the major modes of action of GH47 enzymes.

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 [11]. 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⁺ [12]. 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 [13] and ^3,OB/³S₁ conformations [14]. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry [15], binds GH47 in a ³H₄ conformation [14]. Noeuromycin [14], kifunensine [16] and 1-deoxymannojirimycin [16] all bind in a ¹C₄ conformation, analogous to enzyme-product complexes. Computational studies also support a ^3,OB/³S₁→³H₄→¹C₄ conformational itinerary [14, 17, 18]. 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 [14].

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 [19]. 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 [20, 21]. 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 [13]. 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 [19]. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual ¹C₄ conformation [16]. 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 [22]. 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 [13, 14]. 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 [16]. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad [13]. 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 [23]. The low nanomolar binding of mannoimidazole to CkGH47 is consistent with anti-protonation [14].

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 [19]. 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 [16].

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 [24]. 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 [11].

First general base identification

Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [19]. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in S. cerevisiae) [22].

First general acid identification

Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules [19]. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in S. cerevisiae) [16], however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in S. cerevisiae) [23].

First 3-D structure

Saccharomyces cerevisiae α-1,2-mannosidase [19].

References

1. Herscovics A (2001). Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control. Biochimie. 2001;83(8):757-62. DOI:10.1016/s0300-9084(01)01319-0 |
2. Herscovics A, Romero PA, and Tremblay LO. (2002). The specificity of the yeast and human class I ER alpha 1,2-mannosidases involved in ER quality control is not as strict previously reported. Glycobiology. 2002;12(4):14G-15G. | Google Books | Open Library
3. Tremblay LO and Herscovics A. (2000). Characterization of a cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involved in N-glycan biosynthesis. J Biol Chem. 2000;275(41):31655-60. DOI:10.1074/jbc.M004935200 |
4. Lal A, Pang P, Kalelkar S, Romero PA, Herscovics A, and Moremen KW. (1998). Substrate specificities of recombinant murine Golgi alpha1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases. Glycobiology. 1998;8(10):981-95. DOI:10.1093/glycob/8.10.981 |
5. Hosokawa N, Tremblay LO, Sleno B, Kamiya Y, Wada I, Nagata K, Kato K, and Herscovics A. (2010). EDEM1 accelerates the trimming of alpha1,2-linked mannose on the C branch of N-glycans. Glycobiology. 2010;20(5):567-75. DOI:10.1093/glycob/cwq001 |
6. Hirao K, Natsuka Y, Tamura T, Wada I, Morito D, Natsuka S, Romero P, Sleno B, Tremblay LO, Herscovics A, Nagata K, and Hosokawa N. (2006). EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J Biol Chem. 2006;281(14):9650-8. DOI:10.1074/jbc.M512191200 |
7. Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay LO, Herscovics A, and Nagata K. (2001). A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. EMBO Rep. 2001;2(5):415-22. DOI:10.1093/embo-reports/kve084 |
8. Olivari S, Galli C, Alanen H, Ruddock L, and Molinari M. (2005). A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation. J Biol Chem. 2005;280(4):2424-8. DOI:10.1074/jbc.C400534200 |
9. Lederkremer GZ and Glickman MH. (2005). A window of opportunity: timing protein degradation by trimming of sugars and ubiquitins. Trends Biochem Sci. 2005;30(6):297-303. DOI:10.1016/j.tibs.2005.04.010 |
10. Liebminger E, Hüttner S, Vavra U, Fischl R, Schoberer J, Grass J, Blaukopf C, Seifert GJ, Altmann F, Mach L, and Strasser R. (2009). Class I alpha-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana. Plant Cell. 2009;21(12):3850-67. DOI:10.1105/tpc.109.072363 |
11. Lipari F, Gour-Salin BJ, and Herscovics A. (1995). The Saccharomyces cerevisiae processing alpha 1,2-mannosidase is an inverting glycosidase. Biochem Biophys Res Commun. 1995;209(1):322-6. DOI:10.1006/bbrc.1995.1506 |
12. Jelinek-Kelly S and Herscovics A. (1988). Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc. J Biol Chem. 1988;263(29):14757-63. | Google Books | Open Library
13. Karaveg K, Siriwardena A, Tempel W, Liu ZJ, Glushka J, Wang BC, and Moremen KW. (2005). Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control. J Biol Chem. 2005;280(16):16197-207. DOI:10.1074/jbc.M500119200 |
14. Thompson AJ, Dabin J, Iglesias-Fernández J, Ardèvol A, Dinev Z, Williams SJ, Bande O, Siriwardena A, Moreland C, Hu TC, Smith DK, Gilbert HJ, Rovira C, and Davies GJ. (2012). The reaction coordinate of a bacterial GH47 α-mannosidase: a combined quantum mechanical and structural approach. Angew Chem Int Ed Engl. 2012;51(44):10997-1001. DOI:10.1002/anie.201205338 |
15. Tailford LE, Offen WA, Smith NL, Dumon C, Morland C, Gratien J, Heck MP, Stick RV, Blériot Y, Vasella A, Gilbert HJ, and Davies GJ. (2008). Structural and biochemical evidence for a boat-like transition state in beta-mannosidases. Nat Chem Biol. 2008;4(5):306-12. DOI:10.1038/nchembio.81 |
16. Vallee F, Karaveg K, Herscovics A, Moremen KW, and Howell PL. (2000). Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases. J Biol Chem. 2000;275(52):41287-98. DOI:10.1074/jbc.M006927200 |
17. Mulakala C, Nerinckx W, and Reilly PJ. (2006). Docking studies on glycoside hydrolase Family 47 endoplasmic reticulum alpha-(1-->2)-mannosidase I to elucidate the pathway to the substrate transition state. Carbohydr Res. 2006;341(13):2233-45. DOI:10.1016/j.carres.2006.05.011 |
18. Mulakala C, Nerinckx W, and Reilly PJ. (2007). The fate of beta-D-mannopyranose after its formation by endoplasmic reticulum alpha-(1-->2)-mannosidase I catalysis. Carbohydr Res. 2007;342(2):163-9. DOI:10.1016/j.carres.2006.11.012 |
19. Vallée F, Lipari F, Yip P, Sleno B, Herscovics A, and Howell PL. (2000). Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control. EMBO J. 2000;19(4):581-8. DOI:10.1093/emboj/19.4.581 |
20. Lipari F and Herscovics A. (1999). Calcium binding to the class I alpha-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif. Biochemistry. 1999;38(3):1111-8. DOI:10.1021/bi981643i |
21. Fujita A, Yoshida T, and Ichishima E. (1997). Five crucial carboxyl residues of 1,2-alpha-mannosidase from Aspergillus saitoi (A. phoenicis), a food microorganism, are identified by site-directed mutagenesis. Biochem Biophys Res Commun. 1997;238(3):779-83. DOI:10.1006/bbrc.1997.7389 |
22. Mulakala C and Reilly PJ. (2002). Understanding protein structure-function relationships in Family 47 alpha-1,2-mannosidases through computational docking of ligands. Proteins. 2002;49(1):125-34. DOI:10.1002/prot.10206 |
23. Cantú D, Nerinckx W, and Reilly PJ. (2008). Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum alpha-(1-->2)-mannosidase I. Carbohydr Res. 2008;343(13):2235-42. DOI:10.1016/j.carres.2008.05.026 |
24. Tempel W, Karaveg K, Liu ZJ, Rose J, Wang BC, and Moremen KW. (2004). Structure of mouse Golgi alpha-mannosidase IA reveals the molecular basis for substrate specificity among class 1 (family 47 glycosylhydrolase) alpha1,2-mannosidases. J Biol Chem. 2004;279(28):29774-86. DOI:10.1074/jbc.M403065200 |