Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism

ABSTRACT In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related Nε-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via Nε-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase...

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Autores principales:	Sandy Thao, Jorge C. Escalante-Semerena
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2011
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b7622f655a5e4385ae8da15f59096fc5
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spelling	oai:doaj.org-article:b7622f655a5e4385ae8da15f59096fc52021-11-15T15:38:57ZBiochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism10.1128/mBio.00216-112150-7511https://doaj.org/article/b7622f655a5e4385ae8da15f59096fc52011-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00216-11https://doaj.org/toc/2150-7511ABSTRACT In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related Nε-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via Nε-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, Nε-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, Nε-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that Nε-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by Nε-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.Sandy ThaoJorge C. Escalante-SemerenaAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 2, Iss 5 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Sandy Thao Jorge C. Escalante-Semerena Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
description	ABSTRACT In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related Nε-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via Nε-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, Nε-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, Nε-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that Nε-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by Nε-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.
format	article
author	Sandy Thao Jorge C. Escalante-Semerena
author_facet	Sandy Thao Jorge C. Escalante-Semerena
author_sort	Sandy Thao
title	Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
title_short	Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
title_full	Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
title_fullStr	Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
title_full_unstemmed	Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism
title_sort	biochemical and thermodynamic analyses of <named-content content-type="genus-species">salmonella enterica</named-content> pat, a multidomain, multimeric <italic toggle="yes">n</italic><sup>ε</sup>-lysine acetyltransferase involved in carbon and energy metabolism
publisher	American Society for Microbiology
publishDate	2011
url	https://doaj.org/article/b7622f655a5e4385ae8da15f59096fc5
work_keys_str_mv	AT sandythao biochemicalandthermodynamicanalysesofnamedcontentcontenttypegenusspeciessalmonellaentericanamedcontentpatamultidomainmultimericitalictoggleyesnitalicsupesuplysineacetyltransferaseinvolvedincarbonandenergymetabolism AT jorgecescalantesemerena biochemicalandthermodynamicanalysesofnamedcontentcontenttypegenusspeciessalmonellaentericanamedcontentpatamultidomainmultimericitalictoggleyesnitalicsupesuplysineacetyltransferaseinvolvedincarbonandenergymetabolism
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Biochemical and Thermodynamic Analyses of <named-content content-type="genus-species">Salmonella enterica</named-content> Pat, a Multidomain, Multimeric <italic toggle="yes">N</italic><sup>ε</sup>-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism

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