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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]. These enzymes hydrolyze 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]. Similar trends were seen for Ega3 [2]. Tetraose and longer galactosamine oligosaccharides are hydrolyzed to galactosaminobiose and galactosaminotriose as the final products [7, 8]. Based on the dependence of rate on the chain length of the substrate, it was proposed that the Pseudomonas 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 the catalytic domain 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 [2]. A structural insertion after β-strand 3 creates a deep cleft where galactosamine bound. Ega3 shared 3-D similarity with the family GH166 endo-α-1,4-N-acetylgalactosaminidase Pelh. In silico docking of α-1,4-(GalN)5 revealed six substrate binding subsites.
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].
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. [Tamura1995]
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[Takagi1985]
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. [Tamura1988]
