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

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Escherichia coli YicI is a glycoside hydrolase family (GH) 31 enzyme with alpha-xylosidase activity.
• YicI shares sequence similarity with alpha-glucosidases, suggesting potential for functional conversion.

Purpose of the Study:

• To investigate the structural elements governing YicI substrate specificity.
• To engineer YicI into an alpha-glucosidase through targeted site-directed mutagenesis.

Main Methods:

• Comparative analysis of GH 31 enzyme amino acid sequences.
• Structural comparison of (beta/alpha)(8) barrels in GH 27 and GH 31 enzymes.
• Site-directed mutagenesis of identified key amino acids (Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1) in YicI.

Main Results:

• Specific amino acids and loop regions were identified as crucial for YicI substrate specificity.
• Two YicI mutants, short loop1-enzyme and C307I/F308D, exhibited increased alpha-glucosidase activity.
• The C307I/F308D mutant lost alpha-xylosidase activity and demonstrated successful conversion to an alpha-glucosidase.

Conclusions:

• Targeted mutagenesis of specific residues can alter the substrate specificity of glycoside hydrolases.
• YicI can be successfully engineered into an alpha-glucosidase by modifying key structural elements.