<em>Arabidopsis thaliana</em> aldehyde dehydrogenases ALDH3H1 and ALDH3I1: cysteine mutations, S-nitrosylation and esterase activity

Abstract

S-nitrosylation is a posttranslational modification that is known to affect many biochemical processes in plants. In the current study the ability of NO donors to S-nitrosylate aldehyde dehydrogenases ALDH3H1 and ALDH3I1 from <em>A. thaliana</em> was investigated. It was shown that ALDH3H1 is strongly nitrosylated by GSNO than ALDH3I1 showing the differences in nitrosylating capacity between the two enzymes. The experiments proved that oxidative posttranslational modification modulates the dehydrogenase activity in both enzymes in differential ways. It has been demonstrated that the effect of NO donors is related to the physicochemical properties of S-nitrosoglutathione and sodium nitroprusside. The following conclusions were made from this study. (i) Prolonged incubation time and high dose of NO donors (GSNO and SNP) regulate the inactivation and the rate of dehydrogenase activity. (ii) The decrease of dehydrogenase activity during S-nitrosylation of cysteine residues is related to the loss of sulfhydryl groups. <br /> It is also clearly shown that the reducing reagents DTT and GSH have the differential abilities to restore the GSNO and SNP-mediated inhibition of the dehydrogenase activity in both the enzymes. The effect of the reducing agents on dehydrogenase activity was monitored and it was observed that DTT possessed stronger reducing properties than GSH. The concentration of reducing reagents (DTT and GST) regulated the ability to restore the inactivation of the dehydrogenase activity. Additionally, the dual enzyme activity (dehydrogenase and esterase activities) of aldehyde dehydrogenases ALDH3H1 and ALDH3I1 was effectively demonstrated. Further, the effect of GSNO and CuCl₂ on the esterase activity was investigated and the results indicated that the reactions of dehydrogenation and hydrolysis are probably catalysed at two separate active sites of the protein. Using the non-direct method for detection of S-nitrosylated aldehyde dehydrogenases it is proved that this application can be successfully used in the case of recombinant, purified enzymes ALDH3H1 and ALDH3I1 as well as for ALDH3H1enzyme S-nitrosylated under <em>in vivo</em> conditions. Altogether it can be concluded that ALDH enzymes used in the current study can undergo nitrosylation and apart from having dehydrogenase activity they also possess esterase activity.