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Updated: Jul 12, 2026

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

Modifying the substrate specificity of staphylococcal lipases.

M D van Kampen¹, H M Verheij, M R Egmond

¹Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.

|July 22, 1999

Summary

Researchers identified key residues in Staphylococcus hyicus lipase (SHL) responsible for its high phospholipase activity. Specific mutations, particularly at residue K298, significantly altered both lipase and phospholipase functions, revealing crucial structural determinants.

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Area of Science:

Enzymology
Protein Engineering
Biochemistry

Background:

Staphylococcus hyicus lipase (SHL) exhibits significant phospholipase activity, unlike the homologous Staphylococcus aureus lipase (SAL).
Previous studies identified residues 254-358 in SHL as critical for phospholipid recognition.

Purpose of the Study:

To pinpoint specific amino acid residues within the 254-358 region that dictate SHL's phospholipase activity.
To engineer lipase variants with altered substrate specificities.

Main Methods:

Site-directed mutagenesis was employed, exchanging small clusters of SHL residues (254-358) with their SAL counterparts.
Chimeric enzymes and point mutants were constructed and expressed.
Enzyme kinetics, including k(cat)/K(m) for lipase and phospholipase activities, were analyzed.

Main Results:

One chimera, exchanging SHL residues 293-300 with SAL residues, drastically reduced phospholipase activity (68-fold decrease in k(cat)/K(m)) while increasing lipase activity (32-fold increase).
The K298 residue was identified as a major determinant of phospholipase activity, with the K298F mutation causing a 60-fold decrease in k(cat)/K(m).
Engineering SAL with specific substitutions (e.g., F298K) enhanced its phospholipase activity.

Conclusions:

The region 293-300, particularly residue K298, is crucial for SHL's phospholipase activity.
Modifications in this region can significantly alter the substrate specificity of staphylococcal lipases.
This research provides insights into structure-function relationships for enzyme engineering.