AUTHOR=Potrykus Katarzyna , Thomas Nathan E. , Bruhn-Olszewska Bożena , Sobala Michał , Dylewski Maciej , James Tamara , Cashel Michael 

TITLE=Estimates of RelSeq, Mesh1, and SAHMex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.581271

DOI=10.3389/fmicb.2020.581271

ISSN=1664-302X

ABSTRACT=<p>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<sup>2+</sup> dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from <italic>Drosophila melanogaster</italic> 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<sup>+</sup> 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<sub>Mex</sub>) from <italic>Methylobacterium extorquens</italic> 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 <italic>Streptococcus equisimilis</italic> (Rel<sub>Seq</sub>) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from <italic>M. extorquens</italic> (RSH<sub>Mex</sub>) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.</p>