Characterization of Glucokinases from Pathogenic Free-Living Amoebae

Area of Science:

• Biochemistry
• Molecular Biology
• Parasitology

Background:

• Infections by pathogenic free-living amoebae (FLA) like Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris cause severe central nervous system disease.
• Current treatments for FLA infections are limited, leading to high mortality rates.
• Targeting essential metabolic pathways, such as glucose metabolism, presents a potential therapeutic strategy.

Purpose of the Study:

• To characterize the glucokinase enzymes from Acanthamoeba castellanii (AcGlck) and Balamuthia mandrillaris (BmGlck).
• To compare the kinetic properties of AcGlck and BmGlck with each other and with human glucokinase (HsGlck).
• To assess the potential of AcGlck and BmGlck as targets for novel anti-amoebic therapies.

Main Methods:

• Enzyme kinetics assays were performed to determine kinetic parameters (Km values) for ATP and glucose.
• Apparent Km values for ATP and glucose were measured for AcGlck, BmGlck, and HsGlck.
• Sensitivity of AcGlck and BmGlck to small molecule inhibitors was evaluated.

Main Results:

• AcGlck and BmGlck share 49.3% amino acid identity but exhibit distinct kinetic properties.
• Both AcGlck and BmGlck have significantly higher apparent affinity for glucose compared to HsGlck.
• AcGlck and BmGlck show differential sensitivity to small molecule inhibitors, complicating the development of pan-amoebic drugs.

Conclusions:

• Glucokinases from Acanthamoeba and Balamuthia are viable targets for anti-amoebic drug development.
• Differences in kinetic properties and inhibitor sensitivity between AcGlck, BmGlck, and HsGlck offer opportunities for selective targeting.
• Developing inhibitors with broad activity against multiple FLA glucokinases may be challenging due to enzyme variations.