biostudies-literatureUnknown11Potrykus KEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNarodowe Centrum NaukiThe 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 Mn²⁺ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from <i>Drosophila melanogaster</i> 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 <i>MethFrontiers in microbiology581271https://www.ebi.ac.uk/biostudies/studies/S-EPMC7644958biostudies-literatureEstimates of Rel_Seq, Mesh1, and SAH_Mex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays.PMC7644958Bruhn-Olszewska BDylewski MSobala MCashel MJames TPotrykus KThomas NEfalseEstimates 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 Mn²⁺ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from <i>Drosophila melanogaster</i> 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 <i>Meth2020-01-01T00:00:00Z20202025-04-21T20:27:27.331Z2020-11-22T09:57:57ZS-EPMC76449583319321110.3389/fmicb.2020.581271