ApaH decaps NpN-capped RNAs in two alternative orientations.

Enigmatic dinucleoside tetraphosphates, known as 'alarmones' (NpNs), have recently been shown to function in bacteria as precursors to Np caps on transcripts, likely influencing RNA longevity and cellular adaptation to stress. In proteobacteria, ApaH is the predominant enzyme that hydrolyzes NpNs and decaps Np-capped RNAs to initiate their 5'-end-dependent degradation. Here we conducted a biochemical and structural study to uncover the catalytic mechanism of Escherichia coli ApaH, a prototypic symmetric NpN hydrolase, on various NpNs and Np-capped RNAs. We found that the enzyme uses a unique combination of nonspecific and semispecific substrate recognition, enabling substrates to bind in two orientations with a slight orientational preference. Despite such exceptional recognition properties, ApaH efficiently decaps various Np-capped mRNAs and sRNAs, thereby impacting their lifetimes. Our findings highlight the need to determine substrate orientation preferences before designing substrate-mimicking drugs, as enzymes may escape activity modulation with one of the alternative substrate orientations.