Structural elements to convert Escherichia coli α-xylosidase (YicI) into α-glucosidase

Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an α-xylosidase, although its amino-acid sequence displays approximately 30% identity with α-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (β/α)8 barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and β → α loop 1 of (β/α)8 barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an α-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher α-glucosidase activity than wild-type YicI. C307I/F308D, which lost α-xylosidase activity, was converted into α-glucosidase.