4-Nitrophenyl-α-D-galacturonide

The catalytic activity of the Family 4 glycosidase LplD protein, whose active site motif is CHEV, is unknown despite its crystal structure having been determined in 2008. Here we identify that activity as being an α-galacturonidase whose natural substrate is probably α-1, 4-di-galacturonate (GalUA₂). Phylogenetic analysis shows that LplD belongs to a monophyletic clade of CHEV Family 4 enzymes, of which four other members are also shown to be galacturonidases. Family GH 4 enzymes catalyze the cleavage of the glycosidic bond, via a non-canonical redox-assisted mechanism that contrasts with Koshland’s double-displacement mechanism.

The fungal plant pathogen, Collectotrichum lindemuthianum, was grown in culture with either galactose, arabinose or pectin as the carbon source resulting in the selective secretion of α-galactosidase, α-arabinofuranosidase and exopolygalacturonase, respectively. Each enzyme has been purified by ion exchange and gel permeation chromatography. In addition, a β-glucosidase was purified from the culture filtrate or arabinose grown fungus. The purified α-galactosidase and α-arabinosidase preparations were found to be essentially free of other carbohydrases while the β-glucosidase and exopolygalacturonase preparations contain contaminating activities.

In the first systematic study of the enzyme β-glucuronidase, Masamune showed that oxkidney preparations did not hydrolyse (-)-menthyl α-D-glucuronide, and this compound was also not hydrolysed by mouse-liver β-glucuronidase and by a snail preparation rich in β-glucuronidase and in other simple glycosidases. Nevertheless, although it is not so ubiquitous as the β-glucuronide residue, there is evidence for a naturally occurring α-conjugated glucuronic acid, for example in wheat straw. It has been inferred that uridine diphosphoglucuronic acid also has the α-configuration.