Nuthanakanti Ashok, Korn Megan, Levenson-Palmer Rose, Wu Yue, Babu Nandhini Rajesh, Huang Xuhui, Banh Robert S, Belasco Joel G, Serganov Alexander
Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York City, NY, USA.
Laura and Isaac Perlmutter Cancer Center, New York University Grossman School of Medicine, New York City, NY, USA.
Nat Chem Biol. 2025 Aug 11. doi: 10.1038/s41589-025-01991-4.
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.
神秘的二核苷四磷酸，即所谓的“警报素”（NpNs），最近被证明在细菌中作为转录本上Np帽的前体发挥作用，可能影响RNA寿命和细胞对压力的适应。在变形菌中，ApaH是主要的酶，它水解NpNs并去除Np帽化的RNA的帽，以启动其5'端依赖性降解。在这里，我们进行了一项生化和结构研究，以揭示大肠杆菌ApaH（一种典型的对称NpN水解酶）对各种NpNs和Np帽化RNA的催化机制。我们发现该酶使用非特异性和半特异性底物识别的独特组合，使底物能够以两种方向结合，且有轻微的方向偏好。尽管具有这种特殊的识别特性，ApaH仍能有效地去除各种Np帽化的mRNA和sRNA的帽，从而影响它们的寿命。我们的研究结果强调了在设计模拟底物的药物之前确定底物方向偏好的必要性，因为酶可能会通过其中一种替代底物方向逃避活性调节。
