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

Abstract

An asc operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated from a genomic library in a screen for beta-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 II(asc), while the AscB ORF was highly similar to 6-phospho-beta-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 degrees C, and its activity was enhanced in the presence of Mg(2+) and Ca(2+). The molecular weight of the enzyme was estimated to be 53 000 Da by SDS-PAGE. Two conserved glutamate residues (Glu(182) and Glu(374)) were shown to be important for AscB activity.

• Authors: Chang Long An, Woo Jin Lim, Su Young Hong, Eun Chule Shin, Min Keun Kim, Jong Reoul Lee, Sang Ryeol Park, Jong Gyu Woo, Yong Pyo Lim, Han Dae Yun
• Journal: Research in microbiology (Res Microbiol) Vol. 156 Issue 2 Pg. 145-53 (Mar 2005) ISSN: 0923-2508 [Print] France
• PMID: 15748978 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Phosphoenolpyruvate Sugar Phosphotransferase System
• Glucosidases
• 6-phospho-beta-glucosidase

Topics

• Amino Acid Sequence
• Bacterial Proteins (chemistry, genetics, metabolism)
• Base Sequence
• Cloning, Molecular
• Glucosidases (chemistry, genetics, metabolism)
• Molecular Sequence Data
• Open Reading Frames (genetics)
• Operon
• Pectobacterium carotovorum (enzymology, genetics)
• Phosphoenolpyruvate Sugar Phosphotransferase System (metabolism)
• Sequence Analysis, DNA
• Structure-Activity Relationship