Abstract
Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in bacterial cell growth and division is not well understood. Phosphocholine production by Chok and its attenuation by inhibitors in the context of complex samples such as cell extracts can currently be quantified by several methods. These include choline depletion measurements, radioactive methods, mass-spectrometry, and nuclear magnetic resonance. The first does not measure phosphocholine directly, the second requires elaborate safety procedures, and the third and fourth require significant capital investments and technical expertise. For these reasons, a less expensive, higher throughput, more easily accessible assay is needed to facilitate further study in Gram-positive Choks. Here, we present the development of a triiodide/activated charcoal/molybdenum blue system for detecting and quantifying choline and phosphocholine in parallel. We demonstrate that this system can reliably quantify changes in choline and phosphocholine concentrations over time in Chok enzymatic assays using cell extracts as the source of the enzyme. This is an easily accessible, convenient, robust, and economical method for studying Chok activity in complex samples. The triiodide/activated charcoal/molybdenum blue system opens new doors into the study choline kinase in Gram-positive pathogens.
Highlights
Choline kinase (Chok) is an enzyme that catalyzes the production of phosphocholine (PCho) from choline (Cho) and ATP [1]
Chok enzymes play a key role in cell growth and division in eukaryotic cells [2] and are oncogenic drug targets for cancer cells [3] as well as for parasites such as Plasmodium falciparum [4]
The role Chok plays in bacterial cell division and growth is less clear, it is known to be involved in the pathway leading to the production of lipoteichoic acid (LTA) and cell wall teichoic acid (CTA) [5,6]
Summary
Choline kinase (Chok) is an enzyme that catalyzes the production of phosphocholine (PCho) from choline (Cho) and ATP [1]. Chok enzymes play a key role in cell growth and division in eukaryotic cells [2] and are oncogenic drug targets for cancer cells [3] as well as for parasites such as Plasmodium falciparum [4]. The role Chok plays in bacterial cell division and growth is less clear, it is known to be involved in the pathway leading to the production of lipoteichoic acid (LTA) and cell wall teichoic acid (CTA) [5,6]. Streptococcus pneumoniae is a pathogen known to express Chok [7]. The choline kinase of Streptococcus pneumoniae (sChok) has recently been established as a drug target [8], and inhibiting sChok was found to effectively slow cell growth and division in this species.
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