Molecular characterization of a novel beta-glucuronidase from Scutellaria baicalensis georgi.
We cloned a gene encoding Scutellaria beta-glucuronidase (sGUS) that is involved in the initiation of H(2)O(2) metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing beta-glucuronidase heparanase but no homology to family 2 beta-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 beta-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu(212) or Glu(329) resulted in much lower k(cat)/K(m) values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr(281) is also involved in the beta-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family.
Sasaki
K
K
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
Taura
F
F
Shoyama
Y
Y
Morimoto
S
S
D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D005966:Glucuronidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005966:Glucuronidase