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

Katarzyna Potrykus; Nathan E Thomas; Bożena Bruhn-Olszewska; Michał Sobala; Maciej Dylewski; Tamara James; Michael Cashel

doi:10.3389/fmicb.2020.581271

Estimates of Rel_Seq, Mesh1, and SAH_Mex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

Front Microbiol. 2020 Oct 23:11:581271. doi: 10.3389/fmicb.2020.581271. eCollection 2020.

Authors

Katarzyna Potrykus¹, Nathan E Thomas², Bożena Bruhn-Olszewska¹, Michał Sobala¹, Maciej Dylewski¹, Tamara James², Michael Cashel²

Affiliations

¹ Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland.
² Intramural Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States.

Abstract

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 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.

Keywords: (p)ppApp; (p)ppGpp; Escherichia coli; Mesh1; Methylobacterium extorquens; RSH; SAH; stringent response.