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Estimates of Rel(Seq), Mesh1, and SAH(Mex) Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3′hydroxyl of (p)ppGpp in a Mn(2+) dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the subst...

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Detalles Bibliográficos
Autores principales: Potrykus, Katarzyna, Thomas, Nathan E., Bruhn-Olszewska, Bożena, Sobala, Michał, Dylewski, Maciej, James, Tamara, Cashel, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644958/
https://www.ncbi.nlm.nih.gov/pubmed/33193211
http://dx.doi.org/10.3389/fmicb.2020.581271
Descripción
Sumario:The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3′hydroxyl of (p)ppGpp in a Mn(2+) dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP(+) reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAH(Mex)) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (Rel(Seq)) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSH(Mex)) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.