ApaH decaps Np₄N-capped RNAs in two alternative orientations

Enigmatic dinucleoside tetraphosphates, known as 'alarmones' (Np₄Ns), have recently been shown to function in bacteria as precursors to Np₄ caps on transcripts, likely influencing RNA longevity and cellular adaptation to stress. In proteobacteria, ApaH is the predominant enzyme that hydrolyzes Np₄Ns and decaps Np₄-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 Np₄N hydrolase, on various Np₄Ns and Np₄-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 Np₄-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.