biostudies-literatureBeta RAAUniversity of Thessaly Research CommitteeNational Research FoundationState Scholarships Foundation (IKY) Fellowship of Excellence for Postgraduate Studies in Greece - Siemens Programme (to AK)OMIC-ENGINE (MIS 5002636) implemented under "Reinforcement of the Research and Innovation Infrastructure", Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020)European Regional Development Fund1036-1049https://www.ebi.ac.uk/biostudies/studies/S-EPMC9063446biostudies-literatureUnknown12(5)The 24-h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate-limiting step that determines the lifespan of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site-directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN, we investigated the effect of AMP and neomycin, and used molecular modeling simulations to propose a catalytic mechanism. Collectively, the biochemical and in silico results classify AtHESPERIN in the exonuclease-endonuclease-phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover.FEBS open bioBiochemical and in silico identification of the active site and the catalytic mechanism of the circadian deadenylase HESPERIN.PMC9063446343938175851MIS 5002636Leonidas DDDouka VBalatsos NAAKyritsis ABeta RAAVlachakis DPapanastasi ERizouli MfalseBiochemical and in silico identification of the active site and the catalytic mechanism of the circadian deadenylase HESPERIN.The 24-h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate-limiting step that determines the lifespan of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site-directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN, we investigated the effect of AMP and neomycin, and used molecular modeling simulations to propose a catalytic mechanism. Collectively, the biochemical and in silico results classify AtHESPERIN in the exonuclease-endonuclease-phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover.2022-01-01T00:00:00Z2022 May2026-05-31T06:00:41.759Z2025-02-19T01:55:00.471ZS-EPMC90634463309597710.1002/2211-5463.13011