Glycoside Hydrolase Family 47

2013-01-14T09:52:18Z

Rohan Williams:

{{UnderConstruction}}
* [[Author]]: ^^^Rohan Williams^^^
* [[Responsible Curator]]: ^^^Spencer Williams^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''
|-
|'''Clan'''
|none, (α/α)7 fold
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|debated
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH47.html
|}
</div>


== 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]] <cite>Herscovics2001</cite>. 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 to afford Man8GlcNAc2. Extended incubation results in further demannosylated products ''in vitro'' <cite>Tremblay2002</cite>. 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 <cite>Herscovics2001</cite>.

In mammalian species, the GH47 Golgi mannosidase I (Golgi MI) subfamily acts on Man8-9GlcNAc2 to afford Man5GlcNAc2 and is composed of 3 members (denoted IA, IB and IC) <cite>Herscovics2000</cite>. 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 <cite>Moremen1998</cite>. 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'' <cite>Herscovics2010 Hosokawa2006</cite>. All of these isoforms have been shown to accelerate the disposal of terminally misfolded proteins through ER-associated degradation (ERAD) <cite>Nagata2001 Hosokawa2006 Molinari2005</cite>. 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 <cite>Glickman2005</cite>. It is not clear which mannosidases perform this extensive demannosylation ''in vivo''. Upon extensive demannosylation to Man5-6GlcNAc2 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'' <cite>Strasser2009</cite>.

[[Image:GH47-Figure.png|thumb|center|900px|Schematic depicting the major modes of action of GH47 enzymes.]]

== Kinetics and Mechanism ==
GH47 mannosidases catalyze glycosidic cleavage with [[inverting|inversion]] of stereochemistry, as first determined employing 1H NMR spectroscopy with ''Saccharomyces cervisiae'' α-1,2-mannosidase using Man9GlcNAc as a substrate <cite>Herscovics1995</cite>. 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+ <cite>Herscovics1988</cite>. ''Exo''-α-mannosidases from [[GH38]] and [[GH92]] also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary. Structural studies employing unhydrolysable S-linked substrate analogues have examined the [[Michaelis complex]], with the ligands found to bind in 3''S''1 <cite>Moremen2005</cite> and 3,O''B''/3''S''1 conformations <cite>Davies2012</cite>. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry <cite>Davies2008</cite>, binds GH47 in a 3''H''4 conformation <cite>Davies2012</cite>. Noeuromycin <cite>Davies2012</cite>, kifunensine <cite>HowellJBC2000</cite> and 1-deoxymannojirimycin <cite>HowellJBC2000</cite> all bind in a 1''C''4 conformation, analogous to enzyme-product complexes. Computational studies also support a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Reilly2006 Reilly2007 Davies2012</cite>. 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,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>Herscovics1999 Ischishima1997</cite>. 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 <cite>Moremen2005</cite>. 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 <cite>Howell2000</cite>. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual 1''C''4 conformation <cite>HowellJBC2000</cite>. These complexes were interpreted as being representative of a 1''C''4 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 <cite>Reilly2002</cite>. However, complexes with S-linked substrate analogues implicate a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary, the reverse of that used to preclude Glu330 (Glu132 in ''Saccharomyces'') as the general base residue <cite>Moremen2005 Davies2012</cite>. 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 <cite>HowellJBC2000</cite>. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad <cite>Moremen2005</cite>. 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 <cite>Reilly2008</cite>. The low nanomolar binding of mannoimidazole to ''Ck''GH47 is consistent with ''anti''-protonation <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>HowellJBC2000</cite>.

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 <cite>Moremen2004</cite>. 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 <cite>Herscovics1995</cite>.
;First [[general base]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in ''S. cerevisiae'') <cite>Reilly2002</cite>.
;First [[general acid]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in ''S. cerevisiae'') <cite>HowellJBC2000</cite>, however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in ''S. cerevisiae'') <cite>Reilly2008</cite>.
;First 3-D structure: ''Saccharomyces cerevisiae'' α-1,2-mannosidase <cite>Howell2000</cite>.

== References ==
<biblio>
#Moremen2004 pmid=15102839
#Glickman2005 pmid=15950873
#Strasser2009 pmid=20023195
#Tremblay2002 pmid=12090241
#Nagata2001 pmid=11375934
#Hosokawa2006 pmid=16431915
#Herscovics2010 pmid=20065073
#Molinari2005 pmid=15579471
#Herscovics2000 pmid=10915796
#Moremen1998 pmid=9719679
#Herscovics2001 pmid=11530208
#Ischishima1997 pmid=9325167
#Davies2008 pmid=18408714
#Herscovics1988 pmid=3049586
#Herscovics1999 pmid=9894008
#Herscovics1995 pmid=7726853
#Moremen2005 pmid=15713668
#Davies2012 pmid=23012075
#Reilly2002 pmid=12211022
#Reilly2006 pmid=16806128
#Reilly2007 pmid=17157281
#Reilly2008 pmid=18619586
#HowellJBC2000 pmid=10995765
#Howell2000 pmid=10675327

</biblio>

[[Category:Glycoside Hydrolase Families|GH047]]

Glycoside Hydrolase Family 47

2013-01-14T09:49:28Z

Rohan Williams:

{{UnderConstruction}}
* [[Author]]: ^^^Rohan Williams^^^
* [[Responsible Curator]]: ^^^Spencer Williams^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''
|-
|'''Clan'''
|none, (α/α)7 fold
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|debated
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH47.html
|}
</div>


== 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]] <cite>Herscovics2001</cite>. 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'' <cite>Tremblay2002</cite>. 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 <cite>Herscovics2001</cite>.

In mammalian species, the GH47 Golgi mannosidase I (Golgi MI) subfamily acts on Man8-9GlcNAc2 to afford Man5GlcNAc2 and is composed of 3 members (denoted IA, IB and IC) <cite>Herscovics2000</cite>. 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 <cite>Moremen1998</cite>. 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'' <cite>Herscovics2010 Hosokawa2006</cite>. All of these isoforms have been shown to accelerate the disposal of terminally misfolded proteins through ER-associated degradation (ERAD) <cite>Nagata2001 Hosokawa2006 Molinari2005</cite>. 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 <cite>Glickman2005</cite>. It is not clear which mannosidases perform this extensive demannosylation ''in vivo''. Upon extensive demannosylation to Man5-6GlcNAc2 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'' <cite>Strasser2009</cite>.

[[Image:GH47-Figure.png|thumb|center|900px|Schematic depicting the major modes of action of GH47 enzymes.]]

== Kinetics and Mechanism ==
GH47 mannosidases catalyze glycosidic cleavage with [[inverting|inversion]] of stereochemistry, as first determined employing 1H NMR spectroscopy with ''Saccharomyces cervisiae'' α-1,2-mannosidase using Man9GlcNAc as a substrate <cite>Herscovics1995</cite>. 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+ <cite>Herscovics1988</cite>. ''Exo''-α-mannosidases from [[GH38]] and [[GH92]] also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary. Structural studies employing unhydrolysable S-linked substrate analogues have examined the [[Michaelis complex]], with the ligands found to bind in 3''S''1 <cite>Moremen2005</cite> and 3,O''B''/3''S''1 conformations <cite>Davies2012</cite>. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry <cite>Davies2008</cite>, binds GH47 in a 3''H''4 conformation <cite>Davies2012</cite>. Noeuromycin <cite>Davies2012</cite>, kifunensine <cite>HowellJBC2000</cite> and 1-deoxymannojirimycin <cite>HowellJBC2000</cite> all bind in a 1''C''4 conformation, analogous to enzyme-product complexes. Computational studies also support a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Reilly2006 Reilly2007 Davies2012</cite>. 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,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>Herscovics1999 Ischishima1997</cite>. 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 <cite>Moremen2005</cite>. 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 <cite>Howell2000</cite>. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual 1''C''4 conformation <cite>HowellJBC2000</cite>. These complexes were interpreted as being representative of a 1''C''4 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 <cite>Reilly2002</cite>. However, complexes with S-linked substrate analogues implicate a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary, the reverse of that used to preclude Glu330 (Glu132 in ''Saccharomyces'') as the general base residue <cite>Moremen2005 Davies2012</cite>. 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 <cite>HowellJBC2000</cite>. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad <cite>Moremen2005</cite>. 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 <cite>Reilly2008</cite>. The low nanomolar binding of mannoimidazole to ''Ck''GH47 is consistent with ''anti''-protonation <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>HowellJBC2000</cite>.

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 <cite>Moremen2004</cite>. 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 <cite>Herscovics1995</cite>.
;First [[general base]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in ''S. cerevisiae'') <cite>Reilly2002</cite>.
;First [[general acid]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in ''S. cerevisiae'') <cite>HowellJBC2000</cite>, however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in ''S. cerevisiae'') <cite>Reilly2008</cite>.
;First 3-D structure: ''Saccharomyces cerevisiae'' α-1,2-mannosidase <cite>Howell2000</cite>.

== References ==
<biblio>
#Moremen2004 pmid=15102839
#Glickman2005 pmid=15950873
#Strasser2009 pmid=20023195
#Tremblay2002 pmid=12090241
#Nagata2001 pmid=11375934
#Hosokawa2006 pmid=16431915
#Herscovics2010 pmid=20065073
#Molinari2005 pmid=15579471
#Herscovics2000 pmid=10915796
#Moremen1998 pmid=9719679
#Herscovics2001 pmid=11530208
#Ischishima1997 pmid=9325167
#Davies2008 pmid=18408714
#Herscovics1988 pmid=3049586
#Herscovics1999 pmid=9894008
#Herscovics1995 pmid=7726853
#Moremen2005 pmid=15713668
#Davies2012 pmid=23012075
#Reilly2002 pmid=12211022
#Reilly2006 pmid=16806128
#Reilly2007 pmid=17157281
#Reilly2008 pmid=18619586
#HowellJBC2000 pmid=10995765
#Howell2000 pmid=10675327

</biblio>

[[Category:Glycoside Hydrolase Families|GH047]]

Glycoside Hydrolase Family 47

2013-01-14T09:44:49Z

Rohan Williams:

{{UnderConstruction}}
* [[Author]]: ^^^Rohan Williams^^^
* [[Responsible Curator]]: ^^^Spencer Williams^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''
|-
|'''Clan'''
|none, (α/α)7 fold
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|debated
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH47.html
|}
</div>


== 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]] <cite>Herscovics2001</cite>. 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'' <cite>Tremblay2002</cite>. 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 <cite>Herscovics2001</cite>.

In mammalian species, the GH47 Golgi mannosidase I (Golgi MI) subfamily acts on Man8-9GlcNAc2 to afford Man5GlcNAc2 and is composed of 3 members (denoted IA, IB and IC) <cite>Herscovics2000</cite>. 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 <cite>Moremen1998</cite>. 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'' <cite>Herscovics2010 Hosokawa2006</cite>. All of these isoforms have been shown to accelerate the disposal of terminally misfolded proteins through ER-associated degradation (ERAD) <cite>Nagata2001 Hosokawa2006 Molinari2005</cite>. 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 <cite>Glickman2005</cite>. It is not clear which mannosidases perform this extensive demannosylation ''in vivo''. Upon extensive demannosylation to Man5-6GlcNAc2 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'' <cite>Strasser2009</cite>.

[[Image:GH47-Figure.png|frame|center|3px|Schematic depicting the major modes of action of GH47 enzymes.]]

== Kinetics and Mechanism ==
GH47 mannosidases catalyze glycosidic cleavage with [[inverting|inversion]] of stereochemistry, as first determined employing 1H NMR spectroscopy with ''Saccharomyces cervisiae'' α-1,2-mannosidase using Man9GlcNAc as a substrate <cite>Herscovics1995</cite>. 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+ <cite>Herscovics1988</cite>. ''Exo''-α-mannosidases from [[GH38]] and [[GH92]] also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary. Structural studies employing unhydrolysable S-linked substrate analogues have examined the [[Michaelis complex]], with the ligands found to bind in 3''S''1 <cite>Moremen2005</cite> and 3,O''B''/3''S''1 conformations <cite>Davies2012</cite>. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry <cite>Davies2008</cite>, binds GH47 in a 3''H''4 conformation <cite>Davies2012</cite>. Noeuromycin <cite>Davies2012</cite>, kifunensine <cite>HowellJBC2000</cite> and 1-deoxymannojirimycin <cite>HowellJBC2000</cite> all bind in a 1''C''4 conformation, analogous to enzyme-product complexes. Computational studies also support a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Reilly2006 Reilly2007 Davies2012</cite>. 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,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>Herscovics1999 Ischishima1997</cite>. 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 <cite>Moremen2005</cite>. 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 <cite>Howell2000</cite>. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual 1''C''4 conformation <cite>HowellJBC2000</cite>. These complexes were interpreted as being representative of a 1''C''4 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 <cite>Reilly2002</cite>. However, complexes with S-linked substrate analogues implicate a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary, the reverse of that used to preclude Glu330 (Glu132 in ''Saccharomyces'') as the general base residue <cite>Moremen2005 Davies2012</cite>. 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 <cite>HowellJBC2000</cite>. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad <cite>Moremen2005</cite>. 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 <cite>Reilly2008</cite>. The low nanomolar binding of mannoimidazole to ''Ck''GH47 is consistent with ''anti''-protonation <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>HowellJBC2000</cite>.

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 <cite>Moremen2004</cite>. 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 <cite>Herscovics1995</cite>.
;First [[general base]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in ''S. cerevisiae'') <cite>Reilly2002</cite>.
;First [[general acid]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in ''S. cerevisiae'') <cite>HowellJBC2000</cite>, however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in ''S. cerevisiae'') <cite>Reilly2008</cite>.
;First 3-D structure: ''Saccharomyces cerevisiae'' α-1,2-mannosidase <cite>Howell2000</cite>.

== References ==
<biblio>
#Moremen2004 pmid=15102839
#Glickman2005 pmid=15950873
#Strasser2009 pmid=20023195
#Tremblay2002 pmid=12090241
#Nagata2001 pmid=11375934
#Hosokawa2006 pmid=16431915
#Herscovics2010 pmid=20065073
#Molinari2005 pmid=15579471
#Herscovics2000 pmid=10915796
#Moremen1998 pmid=9719679
#Herscovics2001 pmid=11530208
#Ischishima1997 pmid=9325167
#Davies2008 pmid=18408714
#Herscovics1988 pmid=3049586
#Herscovics1999 pmid=9894008
#Herscovics1995 pmid=7726853
#Moremen2005 pmid=15713668
#Davies2012 pmid=23012075
#Reilly2002 pmid=12211022
#Reilly2006 pmid=16806128
#Reilly2007 pmid=17157281
#Reilly2008 pmid=18619586
#HowellJBC2000 pmid=10995765
#Howell2000 pmid=10675327

</biblio>

[[Category:Glycoside Hydrolase Families|GH047]]

Glycoside Hydrolase Family 47

2013-01-14T09:44:26Z

Rohan Williams:

{{UnderConstruction}}
* [[Author]]: ^^^Rohan Williams^^^
* [[Responsible Curator]]: ^^^Spencer Williams^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''
|-
|'''Clan'''
|none, (α/α)7 fold
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|debated
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH47.html
|}
</div>


== 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]] <cite>Herscovics2001</cite>. 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'' <cite>Tremblay2002</cite>. 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 <cite>Herscovics2001</cite>.

In mammalian species, the GH47 Golgi mannosidase I (Golgi MI) subfamily acts on Man8-9GlcNAc2 to afford Man5GlcNAc2 and is composed of 3 members (denoted IA, IB and IC) <cite>Herscovics2000</cite>. 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 <cite>Moremen1998</cite>. 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'' <cite>Herscovics2010 Hosokawa2006</cite>. All of these isoforms have been shown to accelerate the disposal of terminally misfolded proteins through ER-associated degradation (ERAD) <cite>Nagata2001 Hosokawa2006 Molinari2005</cite>. 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 <cite>Glickman2005</cite>. It is not clear which mannosidases perform this extensive demannosylation ''in vivo''. Upon extensive demannosylation to Man5-6GlcNAc2 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'' <cite>Strasser2009</cite>.

[[Image:GH47-Figure.png|frame|center|{3}px|Schematic depicting the major modes of action of GH47 enzymes.]]

== Kinetics and Mechanism ==
GH47 mannosidases catalyze glycosidic cleavage with [[inverting|inversion]] of stereochemistry, as first determined employing 1H NMR spectroscopy with ''Saccharomyces cervisiae'' α-1,2-mannosidase using Man9GlcNAc as a substrate <cite>Herscovics1995</cite>. 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+ <cite>Herscovics1988</cite>. ''Exo''-α-mannosidases from [[GH38]] and [[GH92]] also require a metal ion for catalysis.

GH47 mannosidases operate through an unusual 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary. Structural studies employing unhydrolysable S-linked substrate analogues have examined the [[Michaelis complex]], with the ligands found to bind in 3''S''1 <cite>Moremen2005</cite> and 3,O''B''/3''S''1 conformations <cite>Davies2012</cite>. Mannoimidazole, whose binding to other mannosidases has been shown to be consistent with good transition state mimicry <cite>Davies2008</cite>, binds GH47 in a 3''H''4 conformation <cite>Davies2012</cite>. Noeuromycin <cite>Davies2012</cite>, kifunensine <cite>HowellJBC2000</cite> and 1-deoxymannojirimycin <cite>HowellJBC2000</cite> all bind in a 1''C''4 conformation, analogous to enzyme-product complexes. Computational studies also support a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Reilly2006 Reilly2007 Davies2012</cite>. 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,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>Herscovics1999 Ischishima1997</cite>. 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 <cite>Moremen2005</cite>. 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 <cite>Howell2000</cite>. Subsequent crystal structures of human ER α-mannosidase I in complex with kifunensine and 1-deoxymannojirimycin bound these ligands in an unusual 1''C''4 conformation <cite>HowellJBC2000</cite>. These complexes were interpreted as being representative of a 1''C''4 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 <cite>Reilly2002</cite>. However, complexes with S-linked substrate analogues implicate a 3,O''B''/3''S''1→3''H''4→1''C''4 conformational itinerary, the reverse of that used to preclude Glu330 (Glu132 in ''Saccharomyces'') as the general base residue <cite>Moremen2005 Davies2012</cite>. 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 <cite>HowellJBC2000</cite>. Arg334 is within ion-pairing distance to Glu330 and coordinates to the same water molecule, suggestive of a possible catalytic zwitterionic arginine-carboxylate dyad <cite>Moremen2005</cite>. 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 <cite>Reilly2008</cite>. The low nanomolar binding of mannoimidazole to ''Ck''GH47 is consistent with ''anti''-protonation <cite>Davies2012</cite>.

== 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 <cite>Howell2000</cite>. 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 <cite>HowellJBC2000</cite>.

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 <cite>Moremen2004</cite>. 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 <cite>Herscovics1995</cite>.
;First [[general base]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Believed to be Glu559 in human ER α-mannosidase I (Glu435 in ''S. cerevisiae'') <cite>Reilly2002</cite>.
;First [[general acid]] identification: Unambiguous identification hindered by presence of 3 carboxylate-containing residues in the active site that coordinate ligands through water molecules <cite>Howell2000</cite>. Reported to be Glu330 in human ER α-mannosidase I (Glu132 in ''S. cerevisiae'') <cite>HowellJBC2000</cite>, however, a computational study has concluded that Asp463 acts as the general acid in human ER α-mannosidase I (Asp275 in ''S. cerevisiae'') <cite>Reilly2008</cite>.
;First 3-D structure: ''Saccharomyces cerevisiae'' α-1,2-mannosidase <cite>Howell2000</cite>.

== References ==
<biblio>
#Moremen2004 pmid=15102839
#Glickman2005 pmid=15950873
#Strasser2009 pmid=20023195
#Tremblay2002 pmid=12090241
#Nagata2001 pmid=11375934
#Hosokawa2006 pmid=16431915
#Herscovics2010 pmid=20065073
#Molinari2005 pmid=15579471
#Herscovics2000 pmid=10915796
#Moremen1998 pmid=9719679
#Herscovics2001 pmid=11530208
#Ischishima1997 pmid=9325167
#Davies2008 pmid=18408714
#Herscovics1988 pmid=3049586
#Herscovics1999 pmid=9894008
#Herscovics1995 pmid=7726853
#Moremen2005 pmid=15713668
#Davies2012 pmid=23012075
#Reilly2002 pmid=12211022
#Reilly2006 pmid=16806128
#Reilly2007 pmid=17157281
#Reilly2008 pmid=18619586
#HowellJBC2000 pmid=10995765
#Howell2000 pmid=10675327

</biblio>

[[Category:Glycoside Hydrolase Families|GH047]]

2013-01-11T00:24:27Z

Rohan Williams: