Functional characterization of COG1713 (YqeK) as a novel diadenosine tetraphosphate hydrolase family

Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular level increases following exposure to various stress signals and stimuli, and its accumulation is generally correlated to increased sensitivity to the stressful condition, decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a byproduct of tRNA aminoacylation, and it is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, starting from the evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically-cleaving Ap4A hydrolases. We validated the functional assignment by confirming YqeK ability to affect the in vivo level of Ap4A in B. subtilis YqeK shows a catalytic efficiency towards Ap4A similar to that of the symmetrically-cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold, typical of proteins of the HD domain superfamily that harbor a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints on substrate binding mode. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, being frequently found in operons involved in an integrated response to stress signals.IMPORTANCEElevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in γ- and β- proteobacteria, and it has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.