Structural and biochemical analysis of the asc operon encoding 6-phospho-β-glucosidase in Pectobacterium carotovorum subsp. carotovorum LY34

An asc operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated from a genomic library in a screen for β-glucosidase activities. Sequence analysis of the 5618-bp cloned DNA fragment (accession number AY622309) showed three open reading frames (ascG, ascF, and ascB) that are predicted to encode 375, 486, and 476 amino acid proteins, respectively. The AscG ORF shared a high similarity with the Escherichia coli AscG repressor. The AscF ORF shared 81% identity with the E. coli AscF PTS enzyme IIasc, while the AscB ORF was highly similar to 6-phospho-β-glucosidases and is a member of the glycosyl hydrolase family 1. The purified AscB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. It exhibited maximal activity at pH 7.0 and 40 °C, and its activity was enhanced in the presence of Mg2+ and Ca2+. The molecular weight of the enzyme was estimated to be 53 000 Da by SDS-PAGE. Two conserved glutamate residues (Glu182 and Glu374) were shown to be important for AscB activity.