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Difference between revisions of "Glycoside Hydrolase Family 67"

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* [[Author]]: [[User:Harry Gilbert|Harry Gilbert]]
 
* [[Author]]: [[User:Harry Gilbert|Harry Gilbert]]
 
* [[Responsible Curator]]:  [[User:Harry Gilbert|Harry Gilbert]]
 
* [[Responsible Curator]]:  [[User:Harry Gilbert|Harry Gilbert]]
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|-
 
|-
 
|'''Clan'''     
 
|'''Clan'''     
|GH-x
+
|none
 
|-
 
|-
 
|'''Mechanism'''
 
|'''Mechanism'''
|retaining/inverting
+
|inverting
 
|-
 
|-
 
|'''Active site residues'''
 
|'''Active site residues'''
|known/not known
+
|Proposed
 
|-
 
|-
 
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 
|-
 
|-
| colspan="2" |http://www.cazy.org/fam/GHnn.html
+
| colspan="2" |{{CAZyDBlink}}GH67.html
 
|}
 
|}
 
</div>
 
</div>
  
 
== Substrate specificities ==
 
== Substrate specificities ==
GH67 contains enzymes that display alpha-glucuronidase activity. The enzymes target the glucuronic acid appended to the C2-OH of the xylose at the non-reducing end of xylooligosaccharides. The enzymes display a preference for 4-O-methyl-D-glucuronic acid side chains. The length of the oligosacchride does not influence catalytic rate indicating that the enzyme only interacts with the uronic acid and the linked xylose. These enzymes do not remove glucuronic acid from internal regions of xylan <cite>#1#2</cite>. The enzymes are generally intracellular or membrane associated <cite>#3#4</cite> suggesting  that they play a terminal role in uncapping decorated xyloooligosacchrides, making these molecules available to beta-xylosidases produced by the host.
+
[[Glycoside hydrolase]]s of this family display alpha-glucuronidase activity. The enzymes target the glucuronic acid appended to the C2-OH of the xylose at the non-reducing end of xylooligosaccharides. The enzymes display a preference for 4-O-methyl-D-glucuronic acid side chains. The length of the oligosaccharide does not influence catalytic rate indicating that the enzyme only interacts with the uronic acid and the linked xylose. These enzymes do not remove glucuronic acid from internal regions of xylan <cite>Ruile1995,Bronnenmeier1995</cite>. The enzymes are generally intracellular or membrane associated <cite>Shulami1999,Nagy2002</cite> suggesting  that they play a terminal role in uncapping decorated xyloooligosaccharides, making these molecules available to beta-xylosidases produced by the host.
  
 
== Kinetics and Mechanism ==
 
== Kinetics and Mechanism ==
Alpha-glucuronidases hydrolyse their target glycoside bond through a single displacement acid-base assisted mechanism, and thus the released glucuronic acid released is in a beta conformation <cite>#5</cite>.
+
&alpha;-glucuronidases are [[inverting]] enzymes that hydrolyse their target glycoside bond through a single displacement mechanism assisted by [[general acid]] and [[general base]] residues. Thus the glucuronic acid is formed in a beta configuration <cite>Biely2000</cite>.
  
 
== Catalytic Residues ==
 
== Catalytic Residues ==
Typical of single displacement glycoside hydrolases, GH67 enzymes contain a catalytic acid that protonates the scissile glycosidic oxygen promoting leaving group departure. This residue, Glu292 in the ''Cellvibrio japonicus'' GH67 <cite>#6</cite> and Glu285 in the ''Geobacillus stearothermophilus'' GH67 enzymes <cite>#7</cite> is a conserved glutamate within GH67. There are a pair of carboxylic acids that make hydrogen bonds with the catalytic water (attacks the anomeric carbon of the scissile glycosidic bond), and are predicted to activate the solvent molecule, thus acting as the catalytic base. Which of these highly conserved residues, Glu393/Asp365 and Glu392/Asp364 in the C. japonicus and G. stearothermophilus enzymes, respectively, act as the catalytic base is unclear. Mutational studies suggested that Asp365 in the ''C. japonicus'' enzyme may be the catalytic base <cite>#6</cite>, although similar mutagenesis studies on the ''Geobacillus'' glucuronidase indicate that mutation of either possible catalytic bases results in almost complete inactivation of the enzyme <cite>#8</cite>.
+
Typical of [[inverting]] glycoside hydrolases, GH67 enzymes contain a [[general acid]] that protonates the scissile glycosidic oxygen promoting leaving group departure. This residue, Glu292 in the ''Cellvibrio japonicus'' GH67 <cite>Nurizzo2002</cite> and Glu285 in the ''Geobacillus stearothermophilus'' GH67 enzymes <cite>Golan2004</cite> is a conserved glutamate within GH67. There are a pair of carboxylic acids that make hydrogen bonds with the catalytic water (attacks the anomeric carbon of the scissile glycosidic bond), and are predicted to activate the solvent molecule, thus acting as the [[general base]]. Which of these highly conserved residues, Glu393/Asp365 and Glu392/Asp364 in the ''C. japonicus'' and ''G. stearothermophilus'' enzymes, respectively, act as the [[general base]] is unclear. Mutational studies suggested that Asp365 in the ''C. japonicus'' enzyme may be the catalytic base <cite>Nurizzo2002</cite>, although similar mutagenesis studies on the ''Geobacillus'' glucuronidase indicate that mutation of either possible catalytic bases results in almost complete inactivation of the enzyme <cite>Zaide2001</cite>.
  
 
== Three-dimensional structures ==
 
== Three-dimensional structures ==
GH67 enzymes contain three distinct domains (cite)#5#6(/cite). The N-terminal domain forms a two-layer β sandwich, the central domain, the catalytic domain, is a classical (β/α)8 barrel whose catalytic center is located on the opposite, “C-terminal” side of the barrel to the N-terminal domain. The remaining, C-terminal domain is mainly α-helical. It wraps around the catalytic domain, making additional interactions both with the N-terminal domain of its parent monomer and also forming the majority of the dimer-surface with the equivalent C-terminal domain of the other monomer of the dimer. The active site comprises a deep, partially hydrophobic, pocket.
+
GH67 enzymes contain three distinct domains <cite>Nurizzo2002,Golan2004</cite>. The N-terminal domain forms a two-layer β sandwich, the central domain, the catalytic domain, is a classical (β/α)8 barrel whose catalytic center is located on the opposite, "C-terminal" side of the barrel to the N-terminal domain. The remaining, C-terminal domain is mainly α-helical. It wraps around the catalytic domain, making additional interactions both with the N-terminal domain of its parent monomer and also forming the majority of the dimer-surface with the equivalent C-terminal domain of the other monomer of the dimer. The active site comprises a deep, partially hydrophobic, pocket.
  
 
== Family Firsts ==
 
== Family Firsts ==
;First sterochemistry determination: Demonstrate by 1H NMR that the released 4-methyl-D-glucuronic acid was a beta anomer and thus the enzyme is an inverter <cite>#8</cite>.
+
;First sterochemistry determination: <sup>1</sup>H NMR demonstrated that the released 4-methyl-D-glucuronic acid was a beta anomer and thus that the enzyme is an inverter <cite>Biely2000</cite>.
;First catalytic nucleophile identification: The catalytic base was suggested by mutagenesis studies only and there remains two potential candidates <cite>#8</cite>
+
;First [[general base]] residue identification: The general base was suggested by mutagenesis studies only and there remains two potential candidates <cite>Zaide2001</cite>
;First general acid/base residue identification: The catalytic acid was suggested by mutagenesis studies only
+
;First [[general acid]] residue identification: The general acid residue was suggested by mutagenesis studies <cite>Zaide2001</cite>
;First 3-D structure: Two reports on the crystal structure of GH67 glucuronidases were published within 18 months of each other <cite>#6#7</cite>
+
;First 3-D structure: Two reports on the crystal structure of GH67 glucuronidases were published within 18 months of each other <cite>Nurizzo2002,Golan2004</cite>
  
 
== References ==
 
== References ==
 
<biblio>
 
<biblio>
#1 pmid=9044261  
+
#Ruile1995 pmid=9044261  
#2 pmid=7496513  
+
#Bronnenmeier1995 pmid=7496513  
#3 pmid=10368143  
+
#Shulami1999 pmid=10368143  
#4 pmid=12169619  
+
#Nagy2002 pmid=12169619  
#5 pmid=10779688
+
#Biely2000 pmid=10779688
#6 pmid=11937059  
+
#Nurizzo2002 pmid=11937059  
#7 pmid=14573597  
+
#Golan2004 pmid=14573597  
#8 pmid=11358519  
+
#Zaide2001 pmid=11358519  
 
</biblio>
 
</biblio>
 +
 +
<!-- DO NOT REMOVE THIS CATEGORY TAG! -->
 +
[[Category:Glycoside Hydrolase Families|GH067]]

Latest revision as of 18:54, 26 February 2018

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Glycoside Hydrolase Family GHnn
Clan none
Mechanism inverting
Active site residues Proposed
CAZy DB link
http://www.cazy.org/GH67.html

Substrate specificities

Glycoside hydrolases of this family display alpha-glucuronidase activity. The enzymes target the glucuronic acid appended to the C2-OH of the xylose at the non-reducing end of xylooligosaccharides. The enzymes display a preference for 4-O-methyl-D-glucuronic acid side chains. The length of the oligosaccharide does not influence catalytic rate indicating that the enzyme only interacts with the uronic acid and the linked xylose. These enzymes do not remove glucuronic acid from internal regions of xylan [1, 2]. The enzymes are generally intracellular or membrane associated [3, 4] suggesting that they play a terminal role in uncapping decorated xyloooligosaccharides, making these molecules available to beta-xylosidases produced by the host.

Kinetics and Mechanism

α-glucuronidases are inverting enzymes that hydrolyse their target glycoside bond through a single displacement mechanism assisted by general acid and general base residues. Thus the glucuronic acid is formed in a beta configuration [5].

Catalytic Residues

Typical of inverting glycoside hydrolases, GH67 enzymes contain a general acid that protonates the scissile glycosidic oxygen promoting leaving group departure. This residue, Glu292 in the Cellvibrio japonicus GH67 [6] and Glu285 in the Geobacillus stearothermophilus GH67 enzymes [7] is a conserved glutamate within GH67. There are a pair of carboxylic acids that make hydrogen bonds with the catalytic water (attacks the anomeric carbon of the scissile glycosidic bond), and are predicted to activate the solvent molecule, thus acting as the general base. Which of these highly conserved residues, Glu393/Asp365 and Glu392/Asp364 in the C. japonicus and G. stearothermophilus enzymes, respectively, act as the general base is unclear. Mutational studies suggested that Asp365 in the C. japonicus enzyme may be the catalytic base [6], although similar mutagenesis studies on the Geobacillus glucuronidase indicate that mutation of either possible catalytic bases results in almost complete inactivation of the enzyme [8].

Three-dimensional structures

GH67 enzymes contain three distinct domains [6, 7]. The N-terminal domain forms a two-layer β sandwich, the central domain, the catalytic domain, is a classical (β/α)8 barrel whose catalytic center is located on the opposite, "C-terminal" side of the barrel to the N-terminal domain. The remaining, C-terminal domain is mainly α-helical. It wraps around the catalytic domain, making additional interactions both with the N-terminal domain of its parent monomer and also forming the majority of the dimer-surface with the equivalent C-terminal domain of the other monomer of the dimer. The active site comprises a deep, partially hydrophobic, pocket.

Family Firsts

First sterochemistry determination
1H NMR demonstrated that the released 4-methyl-D-glucuronic acid was a beta anomer and thus that the enzyme is an inverter [5].
First general base residue identification
The general base was suggested by mutagenesis studies only and there remains two potential candidates [8]
First general acid residue identification
The general acid residue was suggested by mutagenesis studies [8]
First 3-D structure
Two reports on the crystal structure of GH67 glucuronidases were published within 18 months of each other [6, 7]

References

  1. Ruile P, Winterhalter C, and Liebl W. (1997). Isolation and analysis of a gene encoding alpha-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan. Mol Microbiol. 1997;23(2):267-79. DOI:10.1046/j.1365-2958.1997.2011568.x | PubMed ID:9044261 [Ruile1995]
  2. Bronnenmeier K, Meissner H, Stocker S, and Staudenbauer WL. (1995). alpha-D-glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. Microbiology (Reading). 1995;141 ( Pt 9):2033-40. DOI:10.1099/13500872-141-9-2033 | PubMed ID:7496513 [Bronnenmeier1995]
  3. Shulami S, Gat O, Sonenshein AL, and Shoham Y. (1999). The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6. J Bacteriol. 1999;181(12):3695-704. DOI:10.1128/JB.181.12.3695-3704.1999 | PubMed ID:10368143 [Shulami1999]
  4. Nagy T, Emami K, Fontes CM, Ferreira LM, Humphry DR, and Gilbert HJ. (2002). The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan. J Bacteriol. 2002;184(17):4925-9. DOI:10.1128/JB.184.17.4925-4929.2002 | PubMed ID:12169619 [Nagy2002]
  5. Biely P, de Vries RP, Vrsanská M, and Visser J. (2000). Inverting character of alpha-glucuronidase A from Aspergillus tubingensis. Biochim Biophys Acta. 2000;1474(3):360-4. DOI:10.1016/s0304-4165(00)00029-5 | PubMed ID:10779688 [Biely2000]
  6. Nurizzo D, Nagy T, Gilbert HJ, and Davies GJ. (2002). The structural basis for catalysis and specificity of the Pseudomonas cellulosa alpha-glucuronidase, GlcA67A. Structure. 2002;10(4):547-56. DOI:10.1016/s0969-2126(02)00742-6 | PubMed ID:11937059 [Nurizzo2002]
  7. Golan G, Shallom D, Teplitsky A, Zaide G, Shulami S, Baasov T, Stojanoff V, Thompson A, Shoham Y, and Shoham G. (2004). Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications. J Biol Chem. 2004;279(4):3014-24. DOI:10.1074/jbc.M310098200 | PubMed ID:14573597 [Golan2004]
  8. Zaide G, Shallom D, Shulami S, Zolotnitsky G, Golan G, Baasov T, Shoham G, and Shoham Y. (2001). Biochemical characterization and identification of catalytic residues in alpha-glucuronidase from Bacillus stearothermophilus T-6. Eur J Biochem. 2001;268(10):3006-16. DOI:10.1046/j.1432-1327.2001.02193.x | PubMed ID:11358519 [Zaide2001]

All Medline abstracts: PubMed