Aminoaldehyde dehydrogenase 1 from tomato – enzyme structure and possible using as a tool to analyze aldehydes in beverages

Jan Frömmel¹, Martina Kopečná¹, David Kopečný¹, Miroslav Soural², Radka Končitíková¹, Fabio Vianello³ and Marek Šebela¹

¹Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic;
²Department of Organic Chemistry, Faculty of Science, Palacký University, Olomouc, Czech Republic;
³Department of Comparative Biomedicine and Food Science, University of Padua, Italy

Aminoaldehyde dehydrogenases (AMADH, EC 1.2.1.19) belong to the aldehyde dehydrogenase (ALDH) superfamily and oxidize ω-aminoaldehydes to the respective ω-amino acids. First reported X-ray structures of plant aminoaldehyde dehydrogenase were two isoenzymes from pea (Pisum sativum, PsAMADH1 and 2). In this work we focused on characterization of the isoenzyme 1 from tomato (Lycopersicon esulentum, LeAMADH1), which has unusually wide substrate specificity. The enzyme crystals belong to P2₁2₁2₁ space group and contain one dimer per asymmetric unit in line with gel-filtration measurements indicating the enzyme exists as dimer in solution. Each monomer is composed from oligomerization, catalytic and coenzyme binding domains.

Our study on substrate specificity shows, LeAMADH1 oxidizes not only linear aminoaldehydes but a wide range of N-containing heterocyclic aldehydes such as pyridinecarbaldehydes, 3-pyridinylpropanals and aromatic aldehydes too. The K_m values for the best substrates is 10^-4 – 10^-5 M and the V_max/K_m values compared with that for 3-aminopropanal usually was lower than 10%. There were only three substrates (4-pyridinecarbaldehyde, 2-brom-4-pyridinecarbaldehyde and 3-methylthiopropanal) with this value above 10%. By testing several analogues of NAD⁺ coenzyme we observed that good coenzymes are deamino-NAD⁺ and 3-acetylpyridine-NAD⁺ - the activity of LeAMADH1 reached 110% and 75% of those by using NAD⁺ respectively. By using others coenzymes the activity was drastically reduced.

Almost every alcoholic beverage contains several aldehydes which are products of sugar degradation by higher temperatures or as fermentation products. In higher concentrations these aldehydes are not welcomed in spirits because of their toxicity. Many of these aldehydes are substrates of our enzyme, so we tested several samples of spirits – mostly slivovitz – as substrates of LeAMADH1. Its activity usually was around 1% of the activity reached with APAL. In order to create a biosensor for detection of aldehydes in alcoholic beverages we immobilized LeAMADH1 on magnetic nanoparticles and performed first electrochemical measurement by linear sweep voltametry to detect NADH produced by the enzyme reaction.

This work was supported by grant P501/11/1591 from the Czech Science Foundation.