title: A fluorescence-based assay for N-myristoyltransferase activity.
abstract: N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.
authors: Department of Chemistry, Imperial College London, London SW7 2AZ, UK.
authors: Victor
authors: V
authors: Goncalves
authors: James A
authors: JA
authors: Brannigan
authors: Emmanuelle
authors: E
authors: Thinon
authors: Tayo O
authors: TO
authors: Olaleye
authors: Remigiusz
authors: R
authors: Serwa
authors: Salvatore
authors: S
authors: Lanzarone
authors: Anthony J
authors: AJ
authors: Wilkinson
authors: Edward W
authors: EW
authors: Tate
authors: Robin J
authors: RJ
authors: Leatherbarrow
mesh_terms: D000217:Acyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D003065:Coenzyme A; D003374:Coumarins; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009227:Myristic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D009227:Myristic Acids; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D003065:Coenzyme A