S-Nitrosation impairs activity of stress-inducible aldehyde dehydrogenases from Arabidopsis thaliana.

Nitric oxide (NO) is an intracellular messenger that mediates stress responses. Several plant aldehyde dehydrogenase (ALDH) genes are expressed during abiotic stress conditions to reduce the level of cytotoxic aldehydes. We investigated a possible interference between NO and ALDHs, using the isoform ALDH3H1 of Arabidopsis thaliana as model. The physiological NO donor; S-nitrosoglutathione (GSNO), inhibits ALDH3H1 in a time- and concentration-dependent manner. Mutagenesis and ESI-MS/MS analyses show that all Cys residues of ALDH3H1 are targets of GSNO-mediated S-nitrosation. Chemical labelling indicates that the deactivation is due to the conversion of the catalytic thiol into a catalytically non-active nitrosothiol. GSNO has the same effect on the chloroplastic ALDH3I1, suggesting that susceptibility of the catalytic Cys to NO is a common feature of ALDHs. S-Nitrosation and enzymatic inhibition of ALDH were reverted by reducing agents. Our study proves that the function of ALDHs does not exclusively depend on transcriptional regulation, with stress-induced expression, but may be also susceptible to posttranslational regulation through S-nitrosation. We discuss the potential involvement of S-nitrosoglutathione reductase (GSNOR), binding specific cofactors and reducing partners in a protective system of ALDHs in vivo, which will be experimentally corroborated in our forthcoming study.

Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity.

The cofactor-binding site of the NAD(+)-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2'- and 3'-hydroxyl groups of the adenosyl ribose ring of NAD(+) and repels the 2'-phosphate moiety of NADP(+). If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD(+) is altered and rendered the enzyme capable of using NADP(+). This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2'-phosphate of NADP(+). Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP(+). The double mutant ALDH3H1Glu149Thr/Ile200Val showed a good catalysis with NADP(+). Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a "closed" cofactor binding site. The cofactor specificity was shifted even further in favor of NADP(+), as the mutant ALDH3H1E149T/V178R/I200V uses NADP(+) with almost 7-fold higher catalytic efficiency compared to NAD(+). Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.