Xylella fastidiosa esterase rather than hydroxynitrile lyase.

Area of Science:

• Biochemistry
• Enzymology
• Plant Pathology

Background:

• Xylella fastidiosa is a significant plant pathogen affecting crops.
• A previously identified gene, SCJ21.16 (XFa0032), encoded a protein (XfHNL) with presumed hydroxynitrile lyase activity.
• Sequence analysis suggested XfHNL belongs to the FAD-independent hydroxynitrile lyase family, exhibiting an α/β-hydrolase fold.

Purpose of the Study:

• To re-evaluate the enzymatic activity of Xylella fastidiosa protein XfHNL.
• To provide detailed sequence analysis and experimental validation of XfHNL's function.
• To investigate the active site of XfHNL using computational methods.

Main Methods:

• Heterologous expression and purification of XfHNL.
• Enzymatic assays to test mandelonitrile cleavage/synthesis.
• Sequence analysis and homology modeling.
• Ligand docking simulations.

Main Results:

• Purified XfHNL did not catalyze the cleavage or synthesis of mandelonitrile.
• Experimental data confirmed XfHNL functions as a non-enantioselective esterase.
• Homology modeling and docking supported the esterase activity and elucidated active site characteristics.

Conclusions:

• XfHNL is an esterase, not a hydroxynitrile lyase.
• This reclassification provides insights into the evolutionary relationship between esterases and hydroxynitrile lyases.
• Understanding XfHNL's function aids in elucidating the common ancestry of α/β-hydrolase fold enzymes.