Glycoside Hydrolase Family 135

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

Glycoside Hydrolase Family GH135
Clan	none
Mechanism	unknown
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/GH135.html

Substrate specificities

A single report has disclosed fungal glycoside hydrolases with the ability to degrade the fungal heteropolysaccharide galactosaminogalactan (GAG; not to be confused with glycosaminoglycan mucopolysaccharides) [1]. Galactosaminogalactan is produced by Aspergillus fumigatus as a linear heterogeneous exopolysaccharide composed of α-1,4-linked galactose, N-acetylgalactosamine, and galactosamine residues. GAG is found in both a secreted form and bound to the cell wall of hyphae [2]. A recombinant protein from GH135 was demonstrated to hydrolyze both purified and cell wall-associated GAG. While it is unclear precisely where within the GAG chain the enzyme acts [and thus whether it should be considered an α-galactosidase, α-galactosaminase (i.e. cleaving an α-galactosamine linkage, e.g. GH113), or an N-acetyl-α-galactosaminidase], a three-dimensional complex with N-acetylgalactosamine (see below) suggests that the enzyme possesses α-galactosaminidase activity. The majority of genes encoding family 135 members are found in fungi, with smaller numbers in bacteria and archaea.

Kinetics and Mechanism

Very little is known about the kinetics or mechanism of this enzyme, owing to the lack of homogeneous, well-defined substrates. No activity was detected using a range of simple 4-nitrophenyl glycosides [1]. Instead, the cleavage galactosaminogalactan by Sph3 could be monitored by release of reducing ends. It is not known whether the enzyme hydrolyzes substrate with retention or inversion of anomeric configuration.

Catalytic Residues

Structural and sequence alignments identified three conserved acidic amino acid residues, Asp166, Glu167, and Glu222 (numbering for Aspergillus clavatus), which were located within the putative active site groove. Mutagenesis of these residues individually either decreased or abolished the catalytic activity of Sph3 towards purified GAG. The D166A, D166N, and E167A variants displayed no significant activity. The E222A variant displayed no activity, while the E222Q variant retained 60% of wild-type activity.

Three-dimensional structures

Three-dimensional structures are available for Aspergillus clavatus Sph3 [1]. These structures reveal that the protein has a classical (β/α)₈ TIM barrel fold, with two small additional helices, one on either end of the sixth α helix. This fold was described as sharing similarities with enzymes belonging to families GH18, GH27, and GH84. The putative active site was identified through the acquisition of a binary complex of Sph3 with N-acetylgalactosamine. The proposed cleft is lined with highly conserved tyrosine residues and three acidic residues: Asp166, Glu167, and Glu222. In the Sph3-GalNAc complex, the N -acetyl group forms hydrogen bonds with Glu222 and Tyr65. Glu222 is coordinated by Asn202, which in turn hydrogen bonds to Asp166. A hydrogen bond network through a bridging water molecule connects Glu167, Asp166, and the galactopyranose ring oxygen.

Family Firsts

First stereochemistry determination: Unknown.
First catalytic nucleophile identification: Unknown.
First general acid/base residue identification: Unknown.
First 3-D structure: Sph3 from Aspergillus clavatus [1].

References

All Medline abstracts: PubMed