Exploring the active sites of plant aminoaldehyde dehydrogenases using natural and synthetic substrates
Jan Frömmel1, Hana Moskalíková1, Miroslav Soural2, Martina Tylichová1, David Kopečný1 and Marek Šebela1
1Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic.
of Organic Chemistry, Faculty of Science, Palacký University, tř. 17. listopadu
1192/12, CZ-771 46 Olomouc, Czech Republic.
Aminoaldehyde dehydrogenases (AMADH, EC 126.96.36.199) catalyze the terminal step in polyamine catabolism by oxidizing compounds like 4-aminobutyraldehyde (ABAL) and 3-aminopropionaldehyde (APAL). This NAD+-dependent oxidative reaction produces omega-amino acids related to the processes of cellular defense against stress events caused for example by increased drought and salinity (either as direct mediators or precursors). Based on indirect evidences, the enzyme also seems to participate in the production of carnitine in plants. AMADHs belong to the large enzyme superfamily of aldehyde dehydrogenases. In this work, isoenzymes 1 and 2 of plant AMADHs from pea, tomato and maize were obtained as pure recombinant proteins by expression of the respective genes in Escherichia coli followed by affinity purification. There were also eight mutants of the pea AMADH2 available, which had been prepared by site-directed mutagenesis of active-site residues. To characterize the difference in their substrate specificity, the enzymes were subjected to a large kinetic study with natural and synthetic aminoaldehyde and aldehyde substrates. The studied compounds comprised C2-C4 omega-aminoaldehydes and their N-methylated, hydroxy and guanidino analogs, plus n-alkyl aldehydes (C2-C7). For the activity measurements, there were also position isomers available of pyridine-carbaldehyde, 3-pyridinylpropanal and (pyridinylmethyl)lamino aldehydes together with a series of (9H-purin-6-ylamino) aldehydes, (7H-pyrrolo[2,3-d]pyrimidin-4-ylamino) aldehydes and (pyrimidin-2-ylamino) aldehydes. A majority of the studied AMADHs accepted APAL or ABAL as the best substrates and well oxidized their substitution derivatives. One of the mutants of pea AMADH2 behaved as a non-specific aldehyde dehydrogenase oxidizing preferentially capronaldehyde and other n-alkyl or aromatic aldehydes known as less effective substrates for the wild-type enzyme. Another enzyme showed a significant preference for aromatic aldehydes even though ABAL was the best substrate. The crystal structures of pea AMADH1 and AMADH2 together with structural models of the other enzymes were used to discuss the observed differences in substrate properties.
This work was supported by grant 522/08/0555 from the Czech Science Foundation.