A fluorescence-based assay for N-myristoyltransferase activity

Victor Goncalves; James A. Brannigan; Emmanuelle Thinon; Tayo O. Olaleye; Remigiusz Serwa; Salvatore Lanzarone; Anthony J. Wilkinson; Edward W. Tate; Robin J. Leatherbarrow

doi:10.1016/j.ab.2011.10.013

A fluorescence-based assay for N-myristoyltransferase activity

Victor Goncalves, James A. Brannigan, Emmanuelle Thinon, Tayo O. Olaleye, Remigiusz Serwa, Salvatore Lanzarone, Anthony J. Wilkinson, Edward W. Tate, Robin J. Leatherbarrow

Chemistry

Research output: Contribution to journal › Article › peer-review

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. (C) 2011 Elsevier Inc. All rights reserved.

Original language	English
Pages (from-to)	342-344
Number of pages	3
Journal	ANALYTICAL BIOCHEMISTRY
Volume	421
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2011.10.013
Publication status	Published - 1 Feb 2012

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10.1016/j.ab.2011.10.013

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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. (C) 2011 Elsevier Inc. All rights reserved.",

author = "Victor Goncalves and Brannigan, {James A.} and Emmanuelle Thinon and Olaleye, {Tayo O.} and Remigiusz Serwa and Salvatore Lanzarone and Wilkinson, {Anthony J.} and Tate, {Edward W.} and Leatherbarrow, {Robin J.}",

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T1 - A fluorescence-based assay for N-myristoyltransferase activity

AU - Goncalves, Victor

AU - Brannigan, James A.

AU - Thinon, Emmanuelle

AU - Olaleye, Tayo O.

AU - Serwa, Remigiusz

AU - Lanzarone, Salvatore

AU - Wilkinson, Anthony J.

AU - Tate, Edward W.

AU - Leatherbarrow, Robin J.

PY - 2012/2/1

Y1 - 2012/2/1

N2 - 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. (C) 2011 Elsevier Inc. All rights reserved.

AB - 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. (C) 2011 Elsevier Inc. All rights reserved.

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JF - ANALYTICAL BIOCHEMISTRY

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A fluorescence-based assay for N-myristoyltransferase activity

Abstract

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N-Myristol Transferase as a drug target for anti-malarial therapy

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A fluorescence-based assay for N-myristoyltransferase activity

Abstract

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N-Myristol Transferase as a drug target for anti-malarial therapy

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