CAZypedia - User contributions [en-ca]

Glycoside Hydrolase Family 52

2020-09-03T23:19:23Z

Brian Lowrance:

{{UnderConstruction}}
* [[Author]]: ^^^Julie Grondin^^^, ^^^Brian Lowrance^^^
* [[Responsible Curator]]: ^^^Joel Weadge^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH52'''
|-
|'''Clan'''
|GH-O
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH52.html
|}
</div>


== Substrate specificities ==
The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are monospecific, functioning as ''exo''-β-xylosidases (EC [{{EClink}}3.2.1.37 3.2.1.37]) that cleave the terminal xylose residues from the nonreducing end of xylooligosaccharides, such as ''p''NP-β-d-xylopyranoside <cite>Bravman2001, Espina2014</cite>, xylobiose <cite>Espina2014</cite>, xylotriose <cite>Espina2014</cite>. Low levels of α-L-arabinofuranoside activity has also been observed within members of the GH52 family <cite>Suzuki2014, Bravman2003a</cite>, which is similar to the specificity noted for GH13 and GH54 families for β-xylooligosaccharides and α-L-arabinofuranoside. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site <cite>Lee2007, Utt1991</cite>. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity <cite>Huang2014</cite> and transition from a glycosyl hydrolase to a glycosynthase <cite>Dann2014</cite> has been achieved. Some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has been infrequently observed in other glycosyl hydrolases as well <cite>Romero2019</cite>.

== Kinetics and Mechanism ==
Retention of stereochemistry has been observed in GH52 β-xylosidases, characteristic of a classical [[Koshland double-displacement mechanism]] <cite>Koshland1953</cite>. This was first determined by Bravmen, et. al using H-NMR to analyze the breakdown products of ''p''NP-β-D-xylopyranoside by XynB2, a β-xylosidase, from ''Bacillus stearothermophilus'' T-6 <cite>Bravman2001</cite>. The first detailed kinetic analysis within this family was published in 2003 by Bravman, et. al, containing standard kinetics (''p''NP xylobiose kcat/Km= 140 s-1mM-1), pH dependence (enzymatic catalysis is dependent the ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and substrate binding kinetics (xylobiose 17.1x104 M-1, xylotriose 9.6x104 M-1) of the ''exo''-β-xylosidase XynB2 from ''Bacillus stearothermophilus'', among other kinetic parameters <cite>Bravman2003a</cite>.

== Catalytic Residues ==
Site-directed mutagenesis, chemical rescue, and kinetic profiling of XynB2 from ''B. stearothermophilus'' T-6 identified E335 as the [[catalytic nucleophile]], and D495 as the [[general acid/base]] <cite>Bravman2001, Bravman2003b</cite>. The catalytic nucleophile (E335) is conserved within the WVVNEGEY motif, which is found approximately 150 residues up-stream from the EITTYDSLD motif containing the general acid/base (D495). These results were further confirmed following the structural analysis of GH52 from ''Geobacillus thermoglucosidasius'' <cite>Espina2014</cite>, the 6.5Å separation of Glu and Asp in the active site of this enzyme is consistent with other retaining enzymes.

== Three-dimensional structures ==
[[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]

The structure of GH52 consists of an N-terminal β-sandwich domain and a C-terminal (α/α)6 barrel domain, classifying these enzymes into the GH-''O'' clan, similar to that noted for the GH116 family. The exo-acting mode of action of GH52 is reflected in the topology of the active site. The enzyme acts as a dimer in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy 4H3 half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. This mechanism promotes strict ''exo''-β-xylosidase activity while inhibiting activity on large polymers such as xylan.

== Family Firsts ==
;First stereochemistry determination: XynB2 from Bacillus stearothermophilus T-6 by 1H-NMR for the hydrolysis of ''p''NP-β-D-xylopyranoside <cite>Bravman2001</cite>.
;First catalytic nucleophile identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis and chemical rescue <cite>Bravman2003</cite>.
;First general acid/base residue identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis, chemical rescue, and pH profiling <cite>Bravman2003</cite>.
;First 3-D structure: GH52 from Geobacillus thermoglucosidasius NBRC 107763 <cite>Espina2014</cite>.

== References ==

<biblio>
#Bravman2001 pmid=11322943
#Espina2014 pmid=24816105
#Suzuki2001 pmid=11330658
#Bravman2003a pmid=12950180
#Lee2007 pmid=18051350
#Utt1991 pmid=1905520
#Huang2014 pmid=24122394
#Dann2014 pmid=25484225
#Romero2019 pmid=31024890
#Bravman2003b pmid=12738774
#Koshland1953 Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]

</biblio>

[[Category:Glycoside Hydrolase Families|GH052]]

Glycoside Hydrolase Family 52

2020-09-03T21:38:33Z

Brian Lowrance:

{{UnderConstruction}}
* [[Author]]: ^^^Julie Grondin^^^, ^^^Brian Lowrance^^^
* [[Responsible Curator]]:
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH52'''
|-
|'''Clan'''
|GH-O
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH52.html
|}
</div>


== Substrate specificities ==
The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are monospecific, functioning as ''exo''-β-xylosidases (EC [{{EClink}}3.2.1.37 3.2.1.37]) that cleave the terminal xylose residues from the nonreducing end of xylooligosaccharides, such as ''p''NP-β-d-xylopyranoside <cite>Bravman2001, Espina2014</cite>, xylobiose <cite>Espina2014</cite>, xylotriose <cite>Espina2014</cite>. Low levels of α-L-arabinofuranoside activity has also been observed within members of the GH52 family <cite>Suzuki2014, Bravman2003a</cite>, which is similar to the specificity noted for GH13 and GH54 families for β-xylooligosaccharides and α-L-arabinofuranoside. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site <cite>Lee2007, Utt1991</cite>. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity <cite>Huang2014</cite> and transition from a glycosyl hydrolase to a glycosynthase <cite>Dann2014</cite> has been achieved. Some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has been infrequently observed in other glycosyl hydrolases as well <cite>Romero2019</cite>.

== Kinetics and Mechanism ==
Retention of stereochemistry has been observed in GH52 β-xylosidases, characteristic of a classical [[Koshland double-displacement mechanism]] <cite>Koshland1953</cite>. This was first determined by Bravmen, et. al using H-NMR to analyze the breakdown products of ''p''NP-β-D-xylopyranoside by XynB2, a β-xylosidase, from ''Bacillus stearothermophilus'' T-6 <cite>Bravman2001</cite>. The first detailed kinetic analysis within this family was published in 2003 by Bravman, et. al, containing standard kinetics (''p''NP xylobiose kcat/Km= 140 s-1mM-1), pH dependence (enzymatic catalysis is dependent the ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and substrate binding kinetics (xylobiose 17.1x104 M-1, xylotriose 9.6x104 M-1) of the ''exo''-β-xylosidase XynB2 from ''Bacillus stearothermophilus'', among other kinetic parameters <cite>Bravman2003a</cite>.

== Catalytic Residues ==
Site-directed mutagenesis, chemical rescue, and kinetic profiling of XynB2 from ''B. stearothermophilus'' T-6 identified E335 as the [[catalytic nucleophile]], and D495 as the [[general acid/base]] <cite>Bravman2001, Bravman2003b</cite>. The catalytic nucleophile (E335) is conserved within the WVVNEGEY motif, which is found approximately 150 residues up-stream from the EITTYDSLD motif containing the general acid/base (D495). These results were further confirmed following the structural analysis of GH52 from ''Geobacillus thermoglucosidasius'' <cite>Espina2014</cite>, the 6.5Å separation of Glu and Asp in the active site of this enzyme is consistent with other retaining enzymes.

== Three-dimensional structures ==
[[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]

The structure of GH52 consists of an N-terminal β-sandwich domain and a C-terminal (α/α)6 barrel domain, classifying these enzymes into the GH-''O'' clan, similar to that noted for the GH116 family. The exo-acting mode of action of GH52 is reflected in the topology of the active site. The enzyme acts as a dimer in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy 4H3 half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. This mechanism promotes strict ''exo''-β-xylosidase activity while inhibiting activity on large polymers such as xylan.

== Family Firsts ==
;First stereochemistry determination: XynB2 from Bacillus stearothermophilus T-6 by 1H-NMR for the hydrolysis of ''p''NP-β-D-xylopyranoside <cite>Bravman2001</cite>.
;First catalytic nucleophile identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis and chemical rescue <cite>Bravman2003</cite>.
;First general acid/base residue identification: XynB2 from ''Bacillus stearothermophilus'' T-6 by site-directed mutagenesis, chemical rescue, and pH profiling <cite>Bravman2003</cite>.
;First 3-D structure: GH52 from Geobacillus thermoglucosidasius NBRC 107763 <cite>Espina2014</cite>.

== References ==

<biblio>
#Bravman2001 pmid=11322943
#Espina2014 pmid=24816105
#Suzuki2001 pmid=11330658
#Bravman2003a pmid=12950180
#Lee2007 pmid=18051350
#Utt1991 pmid=1905520
#Huang2014 pmid=24122394
#Dann2014 pmid=25484225
#Romero2019 pmid=31024890
#Bravman2003b pmid=12738774
#Koshland1953 Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]

</biblio>

[[Category:Glycoside Hydrolase Families|GH052]]

User:Brian Lowrance

2020-09-03T20:01:02Z

Brian Lowrance:

[[File: Brian Lowrance Graduation Photo.jpg|200px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. ^^^Joel Weadge^^^'s lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of ''Clostridiodes difficile'' [[GH5]] family CcsZ (unpublished).

User:Brian Lowrance

2020-09-03T20:00:41Z

Brian Lowrance:

[[File: Brian Lowrance Graduation Photo.jpg|200px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. ^^^Joel Weadge^^^'s lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation <cite>Bravman2003</cite>and regulation of microbial biofilms, in particular, the study of ''Clostridiodes difficile'' [[GH5]] family CcsZ (unpublished).

<references/>

<biblio>
#Bravman2003 pmid=12950180

</biblio>

User:Brian Lowrance

2020-09-03T19:20:52Z

Brian Lowrance:

User:Brian Lowrance

2020-09-03T19:19:47Z

Brian Lowrance:

User:Brian Lowrance

2020-09-03T19:18:57Z

Brian Lowrance:

User:Brian Lowrance

2020-09-03T18:56:29Z

Brian Lowrance:

User:Brian Lowrance

2020-09-03T18:56:15Z

Brian Lowrance:

[[File: Brian Lowrance Graduation Photo.jpg|200px|right]]

== Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. ^^^Joel Weadge^^^'s lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of ''Clostridiodes difficile'' [[GH5]] family CcsZ (unpublished). ==

User:Brian Lowrance

2020-08-26T14:57:38Z

Brian Lowrance:

[[File: Brian Lowrance Graduation Photo.jpg|200px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

File:Brian Lowrance Graduation Photo.jpg

2020-08-26T14:53:00Z

Brian Lowrance:

User:Brian Lowrance

2020-08-26T14:52:25Z

Brian Lowrance:

[[File: Brian Lowrance Graduation Photo.jpg]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-26T14:50:49Z

Brian Lowrance:

[[File:Brian Lowrance Graduation Photo.jpg|200px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-26T14:50:35Z

Brian Lowrance:

File:Brian Lowrance Graduation Photo.jpg|200px|right

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-26T14:49:37Z

Brian Lowrance:

[[File:Brian Lowrance Graduation Photo.jpg|200px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-26T14:49:17Z

Brian Lowrance:

[[File:Brian Lowrance Graduation Photo.jpg|100px|right]]

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-26T14:48:35Z

Brian Lowrance:

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program as a member of Dr. Joel Weadge's lab. The primary focus of his research is on the characterization of glycosyl hydrolases in relation to their role in the formation and regulation of microbial biofilms, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

[[File:Brian Lowrance Graduation Photo.jpg|100px|right]]

File:Brian Lowrance Graduation Photo.JPG

2020-08-26T14:47:07Z

Brian Lowrance:

User:Brian Lowrance

2020-08-26T14:46:24Z

Brian Lowrance:

User:Brian Lowrance

2020-08-26T14:41:38Z

Brian Lowrance:

User:Brian Lowrance

2020-08-25T19:02:54Z

Brian Lowrance:

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program. The primary focus of this research is on the characterization of glycosyl hydrolases, in particular, the study of Clostridiodes difficile GH5 family CcsZ (unpublished).

User:Brian Lowrance

2020-08-25T18:35:25Z

Brian Lowrance:

Brian Lowrance acquired his BSc in Biology at Wilfrid Laurier University, where he is currently an MSc candidate in the Integrative Biology program, with the primary focus on the characterization of glycosyl hydrolases.