A fluorescence-based assay for N-myristoyltransferase activity

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• N-myristoylation is a crucial protein modification involving the attachment of myristic acid to N-terminal glycine.
• This process is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme found in eukaryotes.
• NMT activity is implicated in the progression of several types of cancer.

Purpose of the Study:

• To develop and validate a sensitive fluorescence-based assay for quantifying human NMT1 and NMT2 enzymatic activity.
• To characterize the expression of NMT1 and NMT2.
• To validate the assay using small molecule inhibitors.

Main Methods:

• A fluorescence-based assay was established utilizing the detection of coenzyme A (CoA) via 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin.
• Human NMT1 and NMT2 were expressed and characterized.
• Assay performance was validated using known small molecule inhibitors.

Main Results:

• A sensitive fluorescence assay for human NMT1 and NMT2 activity was successfully developed.
• The assay allows for the detection of CoA, a byproduct of the NMT reaction.
• Assay validation confirmed its utility with small molecule inhibitors.

Conclusions:

• The developed fluorescence assay provides a sensitive method for studying NMT enzymatic activity.
• This assay is broadly applicable to NMTs across different organisms.
• It serves as a valuable tool for cancer research and drug discovery targeting NMTs.