Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Mandelamide hydrolase (MAH) belongs to the amidase signature family, catalyzing mandelamide hydrolysis.
• While X-ray structures exist for related enzymes, MAH's structure and substrate specificity determinants are less understood.
• The catalytic triad (Ser-cisSer-Lys) is conserved, but substrate recognition residues vary, suggesting potential for specificity modification.

Purpose of the Study:

• To investigate the substrate specificity of MAH beyond mandelamide.
• To identify mutations that alter MAH's substrate preference using a selection method.
• To understand the role of specific residues in substrate recognition and binding pocket dynamics.

Main Methods:

• Assessing MAH's hydrolytic activity on aliphatic substrates like lactamide.
• Developing a selection system to screen for altered mandelamide/lactamide hydrolysis ratios.
• Site-directed mutagenesis and kinetic analysis (kcat/Km) of mutant variants.
• Homology modeling to visualize mutation effects within the putative active site.

Main Results:

• MAH exhibits low-efficiency hydrolysis of lactamide.
• Several mutations were identified that modify substrate binding and preference.
• The Gly202 residue is critical for aromatic substrate preference; G202A and G202V mutations drastically reduce activity on mandelamide.

Conclusions:

• MAH possesses broader substrate capabilities than initially assumed.
• Specific mutations, particularly at Gly202, can significantly re-engineer MAH's substrate specificity.
• These findings provide insights for directed evolution strategies to tailor amidase activity.