Properties of family 79 beta-glucuronidases that hydrolyze beta-glucuronosyl and 4-O-methyl-beta-glucuronosyl residues of arabinogalactan-protein.
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 beta-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native beta-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 beta-GlcA occurred at pH 3.0-4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent Km values of rAnGlcAase were 30.4 microM for PNP beta-GlcA and 422 microM for beta-GlcA-(1-->6)-Gal, and those of rNcGlcAase were 38.3 microM and 378 microM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP beta-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 beta-glucuronidase with the capacity to hydrolyze beta-GlcA and 4-Me-beta-GlcA residues of AGPs, although they differ significantly in their preferences.
Konishi
Tomoyuki
T
Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
Kotake
Toshihisa
T
Soraya
Dina
D
Matsuoka
Koji
K
Koyama
Tetsuo
T
Kaneko
Satoshi
S
Igarashi
Kiyohiko
K
Samejima
Masahiro
M
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002240:Carbohydrate Sequence; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D020719:Glucuronides / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D020719:Glucuronides; D009088:Mucoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C006619:arabinogalactan proteins; D005966:Glucuronidase