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

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

doi:10.3389/fmicb.2020.581271

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 (SAHMex) 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 (RelSeq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSHMex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.

Highlights

Bacterial global regulatory stress responses play a major role in their adaptation to constantly changing environmental conditions
For example in Escherichia coli they directly interact with the RNA polymerase (RNAP) by binding at two distinct sites (Mechold et al, 2013; Ross et al, 2016; Molodtsov et al, 2018), which leads to transcriptional reprogramming allowing for RelSeq, Mesh1, and SAH (p)ppNpp-ase Assays cell survival under harsh conditions
We found pppApp to be synthesized by M. extorquens and E. coli cells in vivo, and RSH protein from M. extorquens (RSHMex) enzyme is the source of both, (p)ppGpp and pppApp in M. extorquens (Sobala et al, 2019)

Summary

Introduction

Bacterial global regulatory stress responses play a major role in their adaptation to constantly changing environmental conditions. One of the best studied responses is the stringent response, characterized by a swift synthesis of large amounts of guanosine tetra- and penta-phosphates, collectively referred to as (p)ppGpp (Potrykus and Cashel, 2008). These second messengers may act through several different modes of action. For example in Escherichia coli they directly interact with the RNA polymerase (RNAP) by binding at two distinct sites (Mechold et al, 2013; Ross et al, 2016; Molodtsov et al, 2018), which leads to transcriptional reprogramming allowing for RelSeq, Mesh, and SAH (p)ppNpp-ase Assays cell survival under harsh conditions. Other putative ppGpp targets have been recently identified in E. coli (Wang et al, 2019)

Methods

Results

Conclusion