Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity

• DOI: 10.1111/tpj.16651
• Publication Date: 2024-02-03T04:51:14Z
• Authors: Maria Meloni; Jacopo Rossi; Silvia Fanti; Giacomo Carloni; Daniele Tedesco; Patrick Treffon; Luca Piccinini; Giuseppe Falini; Paolo Trost; Elizabeth Vierling; Francesco Licausi; Beatrice Giuntoli; Francesco Musiani; Simona Fermani; Mirko Zaffagnini
• Keywords: 0301 basic medicine; 0303 health sciences; Arabidopsis thaliana; Ethanol; Arabidopsis Proteins; 3D structure; Arabidopsis thaliana; alcohol dehydrogenase; cysteine; ethanol; nitrosoglutathione; redox sensitivity; thiol oxidation; zinc ion; Alcohol dehydrogenase; alcohol dehydrogenase; thiol oxidation; Arabidopsis; Alcohol Dehydrogenase; 3D structure; Substrate Specificity; zinc ion; redox sensitivity; S-Nitrosoglutathione; ethanol; Amino Acid Sequence; 3D-structure; nitrosoglutathione; cysteine; Oxidation-Reduction

SUMMARYAlcohol dehydrogenases (ADHs) are a group of zinc‐binding enzymes belonging to the medium‐length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH‐ADH1 and apo‐GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo‐ and holo‐forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long‐chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol‐oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.