Structural and biochemical analysis of the asc operon encoding 6-phospho-beta-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 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.
An
Chang Long
CL
Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, South Korea.
Lim
Woo Jin
WJ
Hong
Su Young
SY
Shin
Eun Chule
EC
Kim
Min Keun
MK
Lee
Jong Reoul
JR
Park
Sang Ryeol
SR
Woo
Jong Gyu
JG
Lim
Yong Pyo
YP
Yun
Han Dae
HD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009876:Operon; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase