Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase |
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Authors: | Okuyama Masayuki Kaneko Akira Mori Haruhide Chiba Seiya Kimura Atsuo |
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Institution: | Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Sapporo 060-8589, Japan. |
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Abstract: | 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. |
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Keywords: | GH glycoside hydrolase family pNP p-nitrophenyl PCR polymerase chain reaction L1Chi chimeric YicI constructed by replacement of loop 1 with that of α-glucosidase RGAL rice α-galactosidase 5FXyl 5-fluoroxylosyl residue |
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