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Difference between revisions of "Glycoside Hydrolase Family 144"

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== Substrate specificities ==
 
== Substrate specificities ==
[[Glycoside hydrolase]] family 144 contains β-1,2-glucan-hydrolyzing enzymes. The characterized enzymes of this family are an ''endo''-β-1,2-glucanase (EC 3.2.1.71) from ''Chitinophaga pinensis'' (CpSGL) and a sophorohydrolase (nonreducing end) (EC 3.2.1.-) from ''Parabacteroides distasonis'' (BDI_3064)  <cite>Abe2017</cite>. CpSGL hydrolyzes β-1,2-glucan and mainly releases β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of 3–5, whereas BDI_3064 efficiently hydrolyzes shorter β-1,2-glucooligosaccarides with DPs of more than 3 to produce sophorose (Glc-β-1,2-Glc) form the nonreducing end of β-1,2-glucooligosaccarides. These enzyme are highly specific for β-1,2-glucan or its shorter oligosaccharides.
+
[[Glycoside hydrolase]] family 144 contains β-1,2-glucan-hydrolyzing enzymes. The characterized enzymes of this family are an ''endo''-β-1,2-glucanase (EC 3.2.1.71) from ''Chitinophaga pinensis'' (CpSGL) and a sophorohydrolase (nonreducing end) (EC 3.2.1.-) from ''Parabacteroides distasonis'' (BDI_3064)  <cite>Abe2017, Shimizu2018</cite>. CpSGL hydrolyzes β-1,2-glucan and mainly releases β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of 3–5, whereas BDI_3064 efficiently hydrolyzes shorter β-1,2-glucooligosaccarides with DPs of more than 3 to produce sophorose (Glc-β-1,2-Glc) from the nonreducing end of β-1,2-glucooligosaccarides. These enzyme are highly specific for β-1,2-glucan or its shorter oligosaccharides.
  
 
== Kinetics and Mechanism ==
 
== Kinetics and Mechanism ==
CpSGL hydrolyzes cyclic β-1,2-glucan, showing that this enzyme is ''endo''-lytic, whereas BDI_3064 does not, indicating that this enzyme is ''exo''-lytic. Monitoring stereochemical course of the hydrolysis of β-1,2-glucan by <sup>1</sup>H-NMR showed that CpSGL use an inverting mechanism to hydrolyze β-1,2-glucan. Monitoring the change of the degree of optical rotation during hydrolysis of β-1,2-glucan by CpSGL also supported this mechanism.
+
CpSGL hydrolyzes cyclic β-1,2-glucan, showing that this enzyme is ''endo''-lytic, whereas BDI_3064 does not, indicating that this enzyme is ''exo''-lytic <cite>Abe2017, Shimizu2018</cite>. Monitoring the stereochemical course of the hydrolysis of β-1,2-glucan by <sup>1</sup>H-NMR showed that CpSGL use an inverting mechanism to hydrolyze β-1,2-glucan <cite>Abe2017</cite>. Monitoring the change of the degree of optical rotation during hydrolysis of β-1,2-glucan by CpSGL also supported this mechanism <cite>Abe2017</cite>.
  
 
== Catalytic Residues ==
 
== Catalytic Residues ==
Mutational analysis of CpSGL indicated that Asp139, Glu142 and Glu211 play important roles in catalysis. However, structural analysis of CpSGL showed that none of these residues are in positions that can directly transfer hydrogen to the O2 atoms of all the glucose moieties in sophorotriose or are proximal to space between the bound glucose and sophorotriose. Comparison of topological positions of the conserved acidic residues in CpSGL with the catalytic residues in inverting GHs with a similar fold (GH8, GH15 and GH162) did not provide clues to the assignment of the catalytic residues. These observation may imply that GH144 enzymes have a non-canonical reaction mechanism.
+
Mutational analysis of CpSGL indicated that Asp139, Glu142, and Glu211 play important roles in catalysis <cite>Abe2017</cite>. However, structural analysis of CpSGL showed that none of these residues are in positions that can directly transfer hydrogen to the O2 atoms of all the glucose moieties in sophorotriose or are proximal to space between the bound glucose and sophorotriose. Comparison of topological positions of the conserved acidic residues in CpSGL with the catalytic residues in inverting GHs with a similar fold (GH8, GH15, and GH162) did not provide clues to the assignment of the catalytic residues. These observation may imply that GH144 enzymes have a non-canonical reaction mechanism.
  
 
== Three-dimensional structures ==
 
== Three-dimensional structures ==
The 3-D structures of CpSGL ([https://www.rcsb.org/structure/5gzh 5GZH] and [https://www.rcsb.org/structure/5gzk 5GZK]) and BDI_3064 ([https://www.rcsb.org/structure/5z06 5Z06]) were determined by X-ray crystallography and showed an (α/α)<sub>6</sub>-fold of this family. BDI_3064 possesses additional N-terminal domains 1 and 2, important for the substrate specificity of this enzyme as described below. The overall structure of CpSGL is similar to that of GH162 ''endo''-β-1,2-glucanase (TfSGL).
+
The 3-D structures of CpSGL ([https://www.rcsb.org/structure/5gzh 5GZH] and [https://www.rcsb.org/structure/5gzk 5GZK]) and BDI_3064 ([https://www.rcsb.org/structure/5z06 5Z06]) were determined by X-ray crystallography and showed an (α/α)<sub>6</sub>-fold of this family <cite>Abe2017</cite>. BDI_3064 possesses additional N-terminal domains 1 and 2, important for the substrate specificity of this enzyme, as described below. The overall structure of CpSGL is similar to that of GH162 ''endo''-β-1,2-glucanase (TfSGL) despite their low sequence similarity <cite>Tanaka2019</cite>.
The crystal structure of CpSGL in complex with glucose and sophorotriose provided the structural basis for substrate recognition of this enzyme. CpSGL possesses the large cleft typical of ''endo''-acting enzymes. HPLC and ESI-MS analyses suggested that the bound glucose and sophorotriose occupies −3 subsite and +1 to +3 subsites, respectively. Docking analysis of CpSGL using sophoropentaose as a ligand supported the subsite assignment. The ligand-free crystal structure and docking analysis of BDI_3064 showed that Arg93 in the N-terminal domain 1 overlaps −3 subsite and completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharides. This structural feature makes BDI_3064 an ''exo''-acting enzyme.
+
The crystal structure of CpSGL in complex with glucose and sophorotriose provided the structural basis for substrate recognition of this enzyme. CpSGL possesses the large cleft typical of ''endo''-acting enzymes. HPLC and ESI-MS analysis suggested that the bound glucose and sophorotriose occupies −3 subsite and +1 to +3 subsites, respectively. Docking analysis of CpSGL using sophoropentaose as a ligand supported the subsite assignment (unpublished data). The ligand-free crystal structure and docking analysis of BDI_3064 showed that Arg93 in the N-terminal domain 1 overlaps −3 subsite and completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharides <cite>Shimizu2018</cite>. This structural feature would make BDI_3064 an ''exo''-acting enzyme.
The unliganded crystal structures of GH144 enzymes from ''Bacteroides'' species ([https://www.rcsb.org/structure/3eu8 3EU8], [https://www.rcsb.org/structure/4gl3 4GL3] and [https://www.rcsb.org/structure/4qt9 4QT9]) were deposited in the PDB database before the deposition of that of CpSGL. However, these ''Bacteroides'' enzymes have not been biochemically characterized.
+
The unliganded crystal structures of GH144 enzymes from ''Bacteroides'' species ([https://www.rcsb.org/structure/3eu8 3EU8], [https://www.rcsb.org/structure/4gl3 4GL3], and [https://www.rcsb.org/structure/4qt9 4QT9]) were deposited in the PDB database before the deposition of that of CpSGL. However, these ''Bacteroides'' enzymes have not been biochemically characterized.
  
 
== Family Firsts ==
 
== Family Firsts ==
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<biblio>
 
<biblio>
 
#Abe2017 pmid=28270506
 
#Abe2017 pmid=28270506
 
+
#Shimizu2018 pmid=29763309
 
+
#Tanaka2019 pmid=30926603
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [http://www.biochemist.org/bio/03004/0026/030040026.pdf Download PDF version].
 
 
</biblio>
 
</biblio>
  
  
 
[[Category:Glycoside Hydrolase Families|GH144]]
 
[[Category:Glycoside Hydrolase Families|GH144]]

Revision as of 02:54, 14 October 2019

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Glycoside Hydrolase Family GH144
Clan None
Mechanism Inverting
Active site residues Not known
CAZy DB link
http://www.cazy.org/GH144.html


Substrate specificities

Glycoside hydrolase family 144 contains β-1,2-glucan-hydrolyzing enzymes. The characterized enzymes of this family are an endo-β-1,2-glucanase (EC 3.2.1.71) from Chitinophaga pinensis (CpSGL) and a sophorohydrolase (nonreducing end) (EC 3.2.1.-) from Parabacteroides distasonis (BDI_3064) [1, 2]. CpSGL hydrolyzes β-1,2-glucan and mainly releases β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of 3–5, whereas BDI_3064 efficiently hydrolyzes shorter β-1,2-glucooligosaccarides with DPs of more than 3 to produce sophorose (Glc-β-1,2-Glc) from the nonreducing end of β-1,2-glucooligosaccarides. These enzyme are highly specific for β-1,2-glucan or its shorter oligosaccharides.

Kinetics and Mechanism

CpSGL hydrolyzes cyclic β-1,2-glucan, showing that this enzyme is endo-lytic, whereas BDI_3064 does not, indicating that this enzyme is exo-lytic [1, 2]. Monitoring the stereochemical course of the hydrolysis of β-1,2-glucan by 1H-NMR showed that CpSGL use an inverting mechanism to hydrolyze β-1,2-glucan [1]. Monitoring the change of the degree of optical rotation during hydrolysis of β-1,2-glucan by CpSGL also supported this mechanism [1].

Catalytic Residues

Mutational analysis of CpSGL indicated that Asp139, Glu142, and Glu211 play important roles in catalysis [1]. However, structural analysis of CpSGL showed that none of these residues are in positions that can directly transfer hydrogen to the O2 atoms of all the glucose moieties in sophorotriose or are proximal to space between the bound glucose and sophorotriose. Comparison of topological positions of the conserved acidic residues in CpSGL with the catalytic residues in inverting GHs with a similar fold (GH8, GH15, and GH162) did not provide clues to the assignment of the catalytic residues. These observation may imply that GH144 enzymes have a non-canonical reaction mechanism.

Three-dimensional structures

The 3-D structures of CpSGL (5GZH and 5GZK) and BDI_3064 (5Z06) were determined by X-ray crystallography and showed an (α/α)6-fold of this family [1]. BDI_3064 possesses additional N-terminal domains 1 and 2, important for the substrate specificity of this enzyme, as described below. The overall structure of CpSGL is similar to that of GH162 endo-β-1,2-glucanase (TfSGL) despite their low sequence similarity [3]. The crystal structure of CpSGL in complex with glucose and sophorotriose provided the structural basis for substrate recognition of this enzyme. CpSGL possesses the large cleft typical of endo-acting enzymes. HPLC and ESI-MS analysis suggested that the bound glucose and sophorotriose occupies −3 subsite and +1 to +3 subsites, respectively. Docking analysis of CpSGL using sophoropentaose as a ligand supported the subsite assignment (unpublished data). The ligand-free crystal structure and docking analysis of BDI_3064 showed that Arg93 in the N-terminal domain 1 overlaps −3 subsite and completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharides [2]. This structural feature would make BDI_3064 an exo-acting enzyme. The unliganded crystal structures of GH144 enzymes from Bacteroides species (3EU8, 4GL3, and 4QT9) were deposited in the PDB database before the deposition of that of CpSGL. However, these Bacteroides enzymes have not been biochemically characterized.

Family Firsts

First stereochemisty determination
Monitoring hydrolysis of β-1,2-glucan by 1H-NMR spectroscopy and polarimetric analysis showed that CpSGL hydrolyzes β-1,2-glucan with inversion of stereochemistry.
First general acid residue identification
Not known.
First general base residue identification
Not known.
First 3-D structure
The first deposited protein in the PDB database is BF9343_0330 protein from Bacteroides fragilis NCTC 9343 (3EU8) followed by the deposition of BACUNI_03963 and BACCAC_03554 proteins from Bacteroides species (4GL3 and 4QT9, respectively). These structures were determined by Joint Center for Structural Genomics in ligand-free form. However, there is no publication about these proteins. The first published structures are those of CpSGL (5GZH and 5GZK), one of which (5GZK) captures sophorotriose and glucose.

References

  1. Abe K, Nakajima M, Yamashita T, Matsunaga H, Kamisuki S, Nihira T, Takahashi Y, Sugimoto N, Miyanaga A, Nakai H, Arakawa T, Fushinobu S, and Taguchi H. (2017) Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family. J Biol Chem. 292, 7487-7506. DOI:10.1074/jbc.M116.762724 | PubMed ID:28270506 | HubMed [Abe2017]
  2. Shimizu H, Nakajima M, Miyanaga A, Takahashi Y, Tanaka N, Kobayashi K, Sugimoto N, Nakai H, and Taguchi H. (2018) Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β-1,2-Glucooligosaccharides from Parabacteroides distasonis. Biochemistry. 57, 3849-3860. DOI:10.1021/acs.biochem.8b00385 | PubMed ID:29763309 | HubMed [Shimizu2018]
  3. Tanaka N, Nakajima M, Narukawa-Nara M, Matsunaga H, Kamisuki S, Aramasa H, Takahashi Y, Sugimoto N, Abe K, Terada T, Miyanaga A, Yamashita T, Sugawara F, Kamakura T, Komba S, Nakai H, and Taguchi H. (2019) Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family. J Biol Chem. 294, 7942-7965. DOI:10.1074/jbc.RA118.007087 | PubMed ID:30926603 | HubMed [Tanaka2019]
All Medline abstracts: PubMed | HubMed