SUMMARY
Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1‐acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N‐AcetylTransferase Activity). Combining kinetics, HPLC‐MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl‐CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6‐acetyl‐Lys, whereas N5‐acetyl‐Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.
Plant aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases and participate in the metabolism of compounds related to amino acids such as polyamines or osmoprotectants. Their broad specificity covers ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The substrate preference of plant AMADHs is determined by the presence of aspartic acid and aromatic residues in the substrate channel. In this work, 15 new N-acyl derivates of 3-aminopropanal (APAL) and 4-aminobutanal (ABAL) were synthesized and confirmed as substrates of two pea AMADH isoenzymes (PsAMADH 1 and 2). The compounds were designed considering the previously demonstrated conversion of N-acetyl derivatives as well as substrate channel dimensions (5-8 Å × 14 Å). The acyl chain length and its branching were found less significant for substrate properties than the length of the initial natural substrate. In general, APAL derivatives were found more efficient than the corresponding ABAL derivatives because of the prevailing higher conversion rates and lower K m values. Differences in enzymatic performance between the two isoenzymes corresponded in part to their preferences to APAL to ABAL. The higher PsAMADH2 affinity to substrates correlated with more frequent occurrence of an excess substrate inhibition. Molecular docking indicated the possible auxiliary role of Tyr163, Ser295 and Gln451 in binding of the new substrates. The only derivative carrying a free carboxyl group (N-adipoyl APAL) was surprisingly better substrate than ABAL in PsAMADH2 reaction indicating that also negatively charged aldehydes might be good substrates for ALDH10 family.
The metabolic degradation of aldehydes is catalyzed by oxidoreductases from which aldehyde dehydrogenases (EC 1.2.1) comprise nonspecific or substrate-specific enzymes. The latter subset is represented, e.g., by NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs; EC 1.2.1.19) oxidizing a group of naturally occurring ω-aminoaldehydes including polyamine oxidation products. Recombinant isoenzymes from pea (PsAMADH1 and 2) and tomato (LeAMADH1 and 2) were subjected to kinetic measurements with synthetic aldehydes containing a nitrogenous heterocycle such as pyridinecarbaldehydes and their halogenated derivatives, (pyridinylmethylamino)-aldehydes, pyridinyl propanals and aldehydes derived from purine, 7-deazapurine and pyrimidine to characterize their substrate specificity and significance of the resulting data for in vivo reactions. The enzymatic production of the corresponding carboxylic acids was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Although the studied AMADHs are largely homologous and supposed to have a very similar active site architecture, significant differences were observed. LeAMADH1 displayed the broadest specificity oxidizing almost all compounds followed by PsAMADH2 and 1. In contrast, LeAMADH2 accepted only a few compounds as substrates. Pyridinyl propanals were converted by all isoenzymes, usually better than pyridinecarbaldehydes and aldehydes with fused rings. The K (m) values for the best substrates were in the range of 10(-5)-10(-4) M. Nevertheless, the catalytic efficiency values (V (max)/K (m)) reached only a very small fraction of that with 3-aminopropanal (except for LeAMADH1 activity with two pyridine-derived compounds). Docking experiments using the crystal structure of PsAMADH2 were involved to discuss differences in results with position isomers or alkyl chain homologs.
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