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

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 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.

Dual role of a (p)ppGpp‐ and (p)ppApp‐degrading enzyme in biofilm formation and interbacterial antagonism

“…These (p)ppApp nucleotides differ from (p)ppGpp only by the presence of an adenine nucleobase instead of guanine. The enzyme Hs MESH1 was demonstrated to possess ppApp‐degrading activity (Jimmy et al., 2020, Potrykus et al., 2020). In a structure of Hs MESH1 bound to NADPH (PDB: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5VXA (Ding et al., 2020)), the dinucleotides’ adenine moiety resided in the same position as the guanine of (p)ppGpp in our model of substrate‐bound Pa SAH, suggesting that Pa SAH might similarly exhibit substrate promiscuity (Figure ).…”

“…We infer from the model of substrate‐bound Pa SAH and our mutational analysis of active site‐lining residues (Figure 3) that the nucleobase of a (p)ppGpp or (p)ppApp substrate would be sandwiched by the side chains of L30 and W143, the latter probably capable of establishing π‐π interactions with the nucleobase and the former roughly occupying the position of an arginine residue commonly found in the HD1 motif of (p)ppGpp hydrolases (Figure 1b). It is unlikely that the (p)ppApp degrading activity of Pa SAH would solely be attributable to the divergence in HD1 because Hs MESH1, containing the canonical arginine, can hydrolyze ppApp (Jimmy et al., 2020, Potrykus et al., 2020). Surprisingly, mutational studies validated the HD1 arginine as indispensable for Rel Tth , Rel Seq , and Hs MESH1 hydrolase activity (Hogg et al., 2004; Sun et al., 2010; Tamman et al., 2020).…”

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

“…21 Recently, Ito and colleagues reported detection of ppGppbut not pppGppin Drosophila and cultured human cells, 22 thus reinvigorating the debate about the exact biological role of MESH1. However, given the preference of MESH1 for pppGpp 20 and the lack of SYNTH-active RSHs in animals, 5 further studies of proposed MESH1-mediated (p)ppGpp metabolism are necessary. In addition to the well-studied (p)ppGpp alarmones, recently the adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) have stepped into the spotlight.…”

“…In addition to the ubiquitous long RSHs, bacteria also encode small monofunctional RSH enzymes that are involved in the control of alarmone nucleotides: small alarmone synthetases (SAS) and small alarmone hydrolases (SAH). , Although no (p)­ppGpp-synthesizing RSHs are encoded in animal genomes, animals do possess an SAH enzyme, the Metazoan SpoT Homologue 1 (MESH1) . While MESH1 can efficiently hydrolyze alarmones in a Mn 2+ -dependent manner, , it was recently proposed to act as a NADPH phosphatase by catalyzing the removal of the 3′-phosphate moiety of NADPH . Recently, Ito and colleagues reported detection of ppGppbut not pppGppin Drosophila and cultured human cells, thus reinvigorating the debate about the exact biological role of MESH1.…”

“…In addition to the well-studied (p)­ppGpp alarmones, recently the adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) have stepped into the spotlight. First, toxic single-domain RSH enzymes were discovered to produce (p)­ppApp to abrogate bacterial growth. , Second, representatives of several SAH subfamiliesincluding MESH1were shown to efficiently hydrolyze (p)­ppApp. ,, …”