Molecular characterization of a novel beta-glucuronidase from Scutellaria baicalensis georgi

We cloned a gene encoding Scutellaria P-glucuronidase (sGUS) that is involv ed in the initiation of H2O2 metabolism in skullcap (Scutellaria baicalensi s). 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 leucin e zipper motif. sGUS has apparent similarity to the heparan sulfate-metabol izing beta-glucuronidase heparanase but no homology to family 2 beta-glucur onidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results sugges ted that sGUS does not belong to the family 2 beta-glucuronidases. We modif ied several residues predicted to act as the acid-base or nucleophilic resi due 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 t he wild-type enzyme, indicating that these are the acid-base and nucleophil ic residues of the active site, respectively. Moreover, similar site-direct ed mutagenesis confirmed that Tyr(281) is also involved in the beta-glucuro nidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various st ructural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family.