Glycoside Hydrolase Family 114

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• Author: ^^^Spencer Williams^^^
• Responsible Curator: ^^^Spencer Williams^^^

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Glycoside Hydrolase Family GH114
Clan	none
Mechanism	retaining
Active site residues	not known
CAZy DB link
http://www.cazy.org/GH114.html

Substrate specificities

Enzymes of glycoside hydrolase family 114 are endo-α-1,4-polygalactosaminidases. Examples include the foundation member of this family from Pseudomonas sp. 881 [1] and Ega3 from Aspergillus fumigatus [2]. This enzyme hydrolyzes α-1,4-polygalactosamine to oligosaccharides in an endo-acting manner. α-1,4-Polygalactosamine, also known as galactosaminoglycan, is a polymer consisting of α-1,4-linked galactosamine residues, which is only partially N-acetylated, and may also contain N-formyl residues. The polysaccharide is biosynthesized by fungi including Aspergillus parasiticus [3] and Paecilomyces sp. I-1 [4]. An endogalactosaminidase has been purified from Streptomyces griseus; the sequence of this protein is unknown [5].

Kinetics and Mechanism

The endo-α-1,4-polygalactosaminidase from Pseudomonas sp. 881 possesses activity on deacetylated α-1,4-polygalactosamine, but has no activity on fully N-acetylated α-1,4-polygalactosamine [6]. Tetraose and longer galactosamine oligosaccharides are hydrolyzed to galactosaminobiose and galactosaminotriose as the final products [7]. Based on the dependence of rate on the chain length of the substrate, it was proposed that the enzyme has 8 subsites [7]. The enzyme is inhibited by metal ions including Hg²⁺, Fe²⁺ and Sn²⁺ [6]. Digest of galactosaminotetraose resulted in the transient formation of galactosaminohexaose through a transglycosylation reaction [7]. This supports the assignment of a retaining mechanism to this enzyme and family, and is consistent with the enzyme utilizing a classical Koshland double-displacement mechanism.

Catalytic Residues

The 3D structure of Ega3 revealed two candidate active site residues: Asp189 and Glu247 [2]. Mutagenesis supports their role as essential catalytic residues. Docking studies of an oligosaccharide led to the proposal that Asp189 is the catalytic nucleophile and Glu247 is the general acid/base, in a classical Koshland retaining mechanism [2].

Three-dimensional structures

The 3D crystal structure of Ega3 revealed a modified (β/α)8-barrel that lacks β-strand 5 and α-helices 1 and 8. A structural insertion after β-strand 3 creates a deep cleft where galactosamine bound.

Family Firsts

First stereochemistry determination

A retaining mechanism may be inferred from report of transglycosylation activity [7].

First catalytic nucleophile identification

Asp189 in Ega3 is proposed to be the catalytic nucleophile [2].

First general acid/base residue identification

Glu247 in Ega3 is proposed to be the catalytic nucleophile [2].

First 3-D structure

Ega3 from Aspergillus fumigatus [2].

References

1. Tamura, J.-I., Hasegawa, K., Kadowaki, K., Igarashi, Y., Kodama, T. Molecular Cloning and Sequence Analysis of the Gene Encoding an Endo a-l,4 Polygalactosaminidase of Pseudomonas sp. 881. J. Fermentation Bioengineer., 1995, 80, 305. DOI: 10.1016/0922-338X(95)94196-X.

2. Bamford NC, Le Mauff F, Subramanian AS, Yip P, Millán C, Zhang Y, Zacharias C, Forman A, Nitz M, Codée JDC, Usón I, Sheppard DC, and Howell PL. (2019). Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms. J Biol Chem. 2019;294(37):13833-13849. DOI:10.1074/jbc.RA119.009910 |
3. DISTLER JJ and ROSEMAN S. (1960). Galactosamine polymers produced by Aspergillus parasiticus. J Biol Chem. 1960;235:2538-41. | Google Books | Open Library

4. Takagi, H., Kadowaki, K. Purification and Chemical Properties of a Flocculant Produced by Paecilomyces. Agric. Biol. Chem. 1985, 49, 3159-3164. DOI: 10.1080/00021369.1985.10867250

5. Reissig JL, Lai WH, and Glasgow JE. (1975). An endogalactosaminidase from Streptomyces griseus. Can J Biochem. 1975;53(12):1237-49. DOI:10.1139/o75-169 |

6. Tamura, J.-I., Takagi, H., Kadowaki, K. Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp., Agric. Biol. Chem. 1988, 52, 2475-2484. DOI: 10.1080/00021369.1988.10869068.

7. Tamura J, Abe T, Hasegawa K, and Kadowaki K. (1992). The Mode of Action of Endo α-1,4 Polygalactosaminidase from Pseudomonas sp. 881 on Galactosaminooligosaccharides. Biosci Biotechnol Biochem. 1992;56(3):380-3. DOI:10.1271/bbb.56.380 |