RRC ID 28584
Author Nakagomi M, Fujimaki N, Ito A, Toda T, Fukasawa H, Shudo K, Tomita R.
Title A novel aromatic carboxylic acid inactivates luciferase by acylation of an enzymatically active regulatory lysine residue.
Journal PLoS ONE
Abstract Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53, reported here for the first time, substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modifies Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53. The insights from this study about the novel inhibitory modification mechanism might help not only to avoid misinterpretation of the results of Luc-based reporter screening assays but also to explain the pharmacological and toxicological effects of carboxylic acid-containing drugs.
Volume 8(9)
Pages e75445
Published 2013
DOI 10.1371/journal.pone.0075445
PII PONE-D-13-26602
PMID 24066181
PMC PMC3774628
MeSH Acylation / drug effects Animals COS Cells Carboxylic Acids / pharmacology* Cell Line Humans Luciferases / chemistry Luciferases / metabolism* Lysine / metabolism Mice Reverse Transcriptase Polymerase Chain Reaction
IF 2.766
Times Cited 0
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pCMVluc+ (RDB03988)