Glycoside Hydrolase Family 67

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• Author: Harry Gilbert
• Responsible Curator: Harry Gilbert

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

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 [1, 2]. 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.

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).

Catalytic Residues

ypical 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)#5(/cite ) and Glu285 in the Geobacillus stearothermophilus GH67 enzymes (cite)#6(/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)#5(/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)#7(/cite ).

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.

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 [3].

First catalytic nucleophile identification

The catalytic base was suggested by mutagenesis studies only and there remains two potential candidates [4, 5, 5, 6, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38]

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 |
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 |