Functional analysis of ISC1 by site-directed mutagenesis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• The yeast Saccharomyces cerevisiae ISC1 gene encodes inositol phosphosphingolipids-phospholipase C, Isc1p, which shares homology with bacterial and mammalian sphingomyelinases.
• Previous work identified Isc1p as a key enzyme in lipid metabolism, but specific catalytic domains remained uncharacterized.

Purpose of the Study:

• To determine the specific amino acid residues and domains within Isc1p that are critical for its catalytic function.
• To investigate the role of a conserved P-loop-like domain in Isc1p activity, Mg2+ binding, and lipid activation.

Main Methods:

• Site-directed mutagenesis was employed to individually mutate conserved amino acid residues (E100, N233, H334) and residues within the P-loop-like domain (G162, D163, G167, K168, S169).
• Enzyme activity assays were performed to assess the catalytic efficiency (Vmax, Km) of wild-type and mutant Isc1p.
• Mg2+ binding affinity (Ka) and activation by phosphatidylserine (PS) were evaluated for the characterized mutants.

Main Results:

• Mutations of E100, N233, and H334 completely abolished Isc1p activity, highlighting their essential catalytic roles.
• Mutations D163A and K168A within the P-loop-like domain also resulted in complete loss of activity.
• Mutations G162A, G167A, and S169A reduced Vmax but maintained similar Km values, indicating the P-loop-like domain is crucial for catalysis and Mg2+ binding, and affects optimal phosphatidylserine activation.

Conclusions:

• The study identifies critical catalytic residues (E100, N233, H334) and a novel P-loop-like domain (residues 162-169) in yeast Isc1p.
• This P-loop-like domain is essential for Isc1p catalysis, Mg2+ binding, and optimal activation by phosphatidylserine.
• These findings provide novel insights into the structure-function relationship of Isc1p and related sphingomyelinases.