Properties of family 79 β-glucuronidases that hydrolyze β-glucuronosyl and 4- O-methyl-β-glucuronosyl residues of arabinogalactan-protein

Abstract

The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, l-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 β-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native β-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP β-GlcA occurred at pH 3.0–4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent K m values of rAnGlcAase were 30.4 μM for PNP β-GlcA and 422 μM for β-GlcA-(1→6)-Gal, and those of rNcGlcAase were 38.3 μM and 378 μM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP β-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of β-glucuronidase with the capacity to hydrolyze β-GlcA and 4-Me-β-GlcA residues of AGPs, although they differ significantly in their preferences.