Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate

Abstract

Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development.

FIGURE 1.

An overview of possible natural substrates oxidized by plant members of the ALDH10 family (AMADHs). APAL is converted to β-alanine, ABAL to GABA, ACAPAL to 3-acetamidopropionate, GBAL to 4-guanidinobutyrate, TMABAL to γ-butyrobetaine, BAL to glycine betaine, and DMSPAL to DMSP. A nomenclature derived from well characterized substrates has been frequently used for individual enzymes: 4-aminobutyraldehyde dehydrogenase (EC 1.2.1.19), 4-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), 4-guanidinobutyraldehyde dehydrogenase (EC 1.2.1.54), and BADH (EC 1.2.1.8).

FIGURE 2.

Relative activities of tomato (A) and maize (B) AMADHs with NAD⁺ analogs. Coenzyme efficiency was measured with 1 mm APAL as a substrate and 500 μm NAD⁺ analogs, in 0.15 m Tris-HCl buffer, pH 9.0. The rate of the NAD⁺-mediated reaction was arbitrarily taken as 100%. Error bars, S.D.

FIGURE 3.

Substrate specificity of ALDH10 isoenzymes from tomato (top) and maize (bottom). The activity of each AMADH isoenzyme with the best substrate was arbitrarily taken as 100%. The measurements were performed with 1 mm substrates in 0.15 m Tris-HCl buffer, pH 9.0, containing 1 mm NAD⁺. The following substrates were tested: APAL, N,N,N-trimethyl-3-aminopropionaldehyde (TMAPAL), 3-guanidinopropionaldehyde (GPAL), ABAL, N,N-dimethyl-4-aminobutyraldehyde (DMABAL), TMABAL, 4-amino-2-hydroxybutyraldehyde (AHBAL), GBAL, 4-guanidino-2-hydroxybutyraldehyde (GHBAL), BAL, acetaldehyde (C2), propionaldehyde (C3), butyraldehyde (C4), valeraldehyde (C5), capronaldehyde (C6), enanthaldehyde (C7), and pyridine carboxaldehydes (PCAL). Specific activity values with 1 mm APAL were 3.1 units mg⁻¹ for SlAMADH1, 3.6 units mg⁻¹ for SlAMADH2, 2.6 units mg⁻¹ for ZmAMADH1a, 1.9 units mg⁻¹ for ZmAMADH1b, and 4.1 units mg⁻¹ for ZmAMADH2. Error bars, S.D.

FIGURE 4.

SlAMADH1 substrate channel with a bound intermediate. A, a close-up view of the active site in subunit A showing a continuous electron density from the F_o − F_c omit map contoured at 3σ between the catalytic Cys-295 and the PEG aldehyde, which forms a thiohemiacetal intermediate. B, a covalent bond is formed between the SG atom of Cys-295 and the C1 atom of the PEG aldehyde. Hydrogen bonds between the intermediate and enzyme residues (Cys-295 and Asn-162) are shown as dashed lines. The angle geometry of this intermediate is shown. C, a close-up view of the active site in subunit B showing a discontinuous electron density map between the catalytic cysteine and the PEG aldehyde. Residues are labeled.

FIGURE 5.

Broad substrate specificity of plant AMADHs (ALDH10 family). A–E, saturation curves for activity determination of ZmAMADHs and SlAMADHs. Data were measured in 0.15 m Tris-HCl buffer, pH 9.0, with APAL, ABAL, TMABAL, GBAL, BAL, 3-PCAL, and 4-PCAL as substrates in the presence of 1 mm NAD⁺. F, relative activity values for all studied AMADHs with ACAPAL and DMSPAL. The data were measured in 0.15 m Tris-HCl, pH 9.0, using 1 mm substrate and in the presence of 1 mm NAD⁺. The activity with APAL was taken as 100%. Error bars, S.D.

FIGURE 6.

Substrate channels of SlAMADH1, ZmAMADH1a, and PsAMADH2. A, a comparison between the substrate channel volumes of SlAMADH1 (in red) and PsAMADH2 (in blue mesh; Protein Data Bank code 3IWJ). The enlarged volume of SlAMADH1 comes from the absence of Trp-109 and Trp-288 and a displacement at position 453; B and C, a transversal volume section of substrate channel of SlAMADH1 (in red) and PsAMADH2 (in blue), both with a docked ABAL ligand. These images show the enlarged middle section of the substrate channel of SlAMADH1, which occurs due to the presence of Ala-289 and Thr-454 residues compared with the presence of Trp-288 and Cys-453 in PsAMADH2. Thr-454 is hydrogen-bonded to Gln-485 from the other subunit and thus is involved in the dimer interface. Amino acid residues are shown as sticks and labeled. The total surface of the cavities was calculated using Hollow with 0.5-Å grid spacing and a 1.4-Å interior probe. The ABAL molecule (in black) is shown for illustration. It was docked into the active site using AutoDock, and its carbonyl oxygen atom establishes a hydrogen bond to the catalytic cysteine Cys-295/Cys-294, whereas the amino group is hydrogen-bonded to Tyr-163/Tyr-163; D and E, a substrate channel structural comparison of ZmAMADH1a (in purple) and SlAMADH1 (in orange) with PsAMADH2 (in blue, used as the reference for residue numbering shown in parentheses). Cys-446 in ZmAMADH1a allows a conserved Trp to move away from Tyr-165, thus opening the channel for a bulkier BAL molecule. Asn-290 in SlAMADH1 could have a similar role on the opposite side, allowing Tyr-163 to move toward Asn-290, thus opening the channel. Residues labeled in red are not totally conserved in plant AMADHs.

FIGURE 7.

Expression profiles of two SlAMADH and three ZmAMADH genes in various organs as evaluated by qPCR. Transcript abundance is expressed as gene copy number in 1 ng of total RNA amplified by qPCR, normalized to EF1α, and recalculated as primer pair efficiency. Tomato samples, including roots and cotyledons, were collected at 10 days after germination (DAG); shoot apices (stem plus leaves), stems below flowers (including sepals), stems below fruits, petals, reproductive organs, green fruit, roots, and senescent leaves were collected from 6-week-old plants grown on a soil/peat mixture (2:1, v/v). Maize samples were collected from 3-month-old plants unless indicated otherwise. Tassels, silks, and kernels were collected at several days before, during, and after pollination (DAP). Error bars, S.D.

FIGURE 8.

Phylogenetic analysis of the plant ALDH10 family (AMADHs). The unrooted phylogenetic consensus tree shows two distinct groups of AMADHs, one from dicots (solid line) and the other from monocots (dashed line). PsAMADH numbering is used to point out crucial residues modulating substrate specificity. AMADHs exhibiting a significant BADH activity are highlighted in green. AMADHs predictable as broad substrate specific enzymes are highlighted in yellow due to the presence of Thr-454. The presence of Asn-289 implies that Tyr-163 is mobile, affecting the substrate binding. The enzymes from maize (ZmAMADHs) and tomato (SlAMADHs) analyzed in this work are shown in boldface type, as are the previously characterized reference enzymes from peas (PsAMADHs) (4), rice (OsAMADHs) (6), and barley (HvAMADHs) (18). The internal labels give bootstrap frequencies for each clade. See “Experimental Procedures” for the corresponding GenBank^TM accession numbers.