Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase.
Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.
Okuyama
Masayuki
M
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Sapporo 060-8589, Japan.
Kaneko
Akira
A
Mori
Haruhide
H
Chiba
Seiya
S
Kimura
Atsuo
A
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; C503200:YicI protein, E coli; D000520:alpha-Glucosidases