Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues

The first primary structure of a plant aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) is reported. The enzyme of pea (Pisum sativum) seedlings subjected to our study oxidises ω-aminoaldehydes to the corresponding ω-amino acids. Although pea does not accumulate betaine aldehyde as a compatible osmolyte, the N-terminal sequence of a purified pea AMADH resembles those of plant betaine aldehyde dehydrogenases (BADHs). On the basis of an anticipated pea AMADH homology to these enzymes, degenerated oligonucleotide primers were designed and used for PCR amplification. Two cDNA fragments were obtained in initial 5′ RACE experiments. Subsequent 5′and 3′ RACE performed with specific non-degenerated primers provided two putative cDNAs of the plant BADH family. Both encoded protein sequences (AMADH1 and AMADH2) are highly homologous to those of plant BADHs. They show 81% identity and 92% in mutual alignment. As a deduced product of the first cDNA, AMADH1 completely matches the N-terminal sequence of pea AMADH analysed previously by Edman degradation. AMADH 2 represents a putative AMADH or BADH that has not yet been isolated and characterised. We also tried to identify essential amino acid residues of a purified pea AMADH by both determination of its dissociation constants and evaluation of inhibition effects of specific modification reagents. From our results, it is clear that there are Cys (pK = 8.0) and Glu/Asp residues at the active site participating in the catalysis. This is in accordance with the presence of the conserved Glu and Cys active site regions of plant BADHs in both AMADH1 and AMADH2.