Phospholipid synthesizing enzymes of dermatophytes: II. Characterization of choline kinase

Area of Science:

• Biochemistry
• Mycology
• Enzymology

Background:

• Choline kinase (EC 2.7.1.83) is a key enzyme in phospholipid biosynthesis.
• Dermatophytes like Microsporum gypseum and Epidermophyton floccosum are pathogenic fungi causing skin infections.

Purpose of the Study:

• To investigate and compare the biochemical properties of choline kinase from two pathogenic dermatophytes, M. gypseum and E. floccosum.
• To understand the enzyme's substrate specificity, optimal conditions, and inhibition patterns.

Main Methods:

• Enzyme extraction from cytosolic fractions of M. gypseum and E. floccosum.
• Enzyme activity assays across various pH levels and substrate concentrations.
• Determination of kinetic parameters (Km) for choline and ATP.
• Investigation of enzyme inhibition by metal ions and reaction products.

Main Results:

• Choline kinase was localized in the cytosolic fractions of both fungi.
• M. gypseum enzyme exhibited broad pH optima (6.0-9.0), while E. floccosum enzyme showed activity at pH 8.4 and 10.5 (higher at 8.4).
• Both enzymes had a Km of 3.3×10(-4) M for choline. ATP Km values were 6.6×10(-4) M (M. gypseum) and 12.6×10(-4) M (E. floccosum).
• SH-group requirement was observed for both enzymes.
• M. gypseum choline kinase was more sensitive to Mn(2+), Ca(2+), and Ba(2+) inhibition than the E. floccosum enzyme.
• ATP was the preferred phosphate donor, but high concentrations inhibited both enzymes.
• Phosphorylcholine inhibited activity, while phosphoethanolamine and glycerol 3-phosphate were stimulatory.

Conclusions:

• Choline kinase from M. gypseum and E. floccosum share some properties but exhibit distinct characteristics in pH optima and metal ion sensitivity.
• These findings provide insights into the enzymatic machinery of dermatophytes, potentially relevant for antifungal drug development targeting phospholipid metabolism.