Molecular insights into the promiscuous Ap₄N hydrolase YqeK

Diadenosine tetraphosphate (Ap₄A) and related dinucleoside tetraphosphates (Ap₄Ns) are important stress-signaling molecules that coordinate bacterial adaptation to changing environmental conditions. Although the enzymes for turnover of Ap₄A are known in several bacteria, the structural basis for substrate recognition and the cellular consequences of impaired Ap₄A turnover remain poorly understood. Here, we characterize the Histidine-Aspartate (HD)-domain hydrolase YqeK from Bacillus subtilis. Deletion of yqeK impaired growth in stationary phase, sporulation, and biofilm formation, demonstrating a general role under nutrient limitation. YqeK forms a homodimer and functions as a manganese-dependent phosphohydrolase, symmetrically cleaving Ap₄A into two ADP molecules and removing Ap₄A caps from RNA. The enzyme was active not only toward Ap₄A but also toward the mixed dinucleotides Ap₄G, Ap₄C, and Ap₄U in both in vitro and in vivo assays, hence acting as a broad-spectrum regulator of Ap₄N homeostasis. To understand this promiscuity, we determined crystal structures of YqeK in its apo- and ADP-bound state and in complex with a non-hydrolysable Ap₄A analogue. The structures revealed an asymmetric recognition mechanism in which one nucleoside moiety and the proximal phosphate groups are tightly coordinated, whereas the distal nucleoside is accommodated largely through nonspecific interactions, explaining the ability of YqeK to process diverse substrates. Together, our findings establish YqeK as a central regulator of dinucleotide homeostasis and RNA metabolism and provide the structural framework for Ap₄N recognition by HD-domain phosphohydrolases.