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

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.

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum.

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD(+) as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD(+), the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD(+) and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.

Plant aminoaldehyde dehydrogenases oxidize a wide range of nitrogenous heterocyclic aldehydes.

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.

Carboxylate and aromatic active-site residues are determinants of high-affinity binding of ω-aminoaldehydes to plant aminoaldehyde dehydrogenases.

The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.

Biochemical characterization of pea ornithine-delta-aminotransferase: substrate specificity and inhibition by di- and polyamines.

Ornithine-delta-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of L-ornithine to L-glutamate-gamma-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5'-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. L-Ornithine was the best substrate (K(m)=15 mM) but PsOAT also slowly converted N(alpha)-acetyl-L-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K(m)=2mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.

Low molecular weight glutenins in wheat-dependant, exercise-induced anaphylaxis: allergenicity and antigenic relationships with omega 5-gliadins.

BACKGROUND: Adults suffering from wheat-dependant, exercise-induced anaphylaxis (WDEIA) develop IgE directed against wheat omega5-gliadins (major allergens for this allergy) and against wheat low-molecular weight glutenin subunits (LMW-GS). However, the ability of LMW-GS to trigger an inflammatory response is still unknown. It also remains to be determined if IgE from these patients bind the same epitopes on LMW-GS and omega5-gliadins or if the epitopes are independent. METHODS: WDEIA patients were selected and skin prick tests (SPTs) were performed on them using commercial gluten, wheat flour extracts, prolamin fractions and a purified natural LMW-GS P42. The IgE-binding ability of natural and recombinant wheat prolamins was verified by immunoblot experiments. Cross-reactivity between LMW-GS and omega5-gliadins was studied by immunoblot inhibition experiments, using purified natural omega5-gliadin as an inhibitor. RESULTS: Patients developed positive SPTs with natural LMW-GS fractions and/or with the purified LMW-GS P42. Natural and recombinant LMW-GS were highly reactive with patient IgE in immunoblot experiments, as was omega5-gliadin. However, differences in reactivity were evident within the LMW-GS group. Except for one recombinant LMW-GS (P73), IgE cross-reactivity between LMW-GS and natural omega5-gliadin was only partial. CONCLUSION: LMW-GS are able to promote local inflammation and they share common epitopes with omega5-gliadins. The nature of these epitopes is discussed. LMW-GS also carried specific epitopes, completely independent from the omega5-gliadin epitopes. Thus, LMW-GS behaved partly as independent allergens.

Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes.

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived omega-aminoaldehydes to the corresponding omega-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with beta-nicotinamide adenine dinucleotide (NAD(+)) at 2.4 and 2.15 A resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD(+) as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD(+) binding site. While the NAD(+) binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into gamma-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, beta-alanine betaine and gamma-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.

Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum.

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Aminoaldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 A resolution at 100 K. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 A, beta = 98.1 degrees. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit.

Ornithine delta-aminotransferase: An enzyme implicated in salt tolerance in higher plants.

This review deals with biochemical and physiological aspects of plant ornithine d-aminotransferase (OAT, EC 2.6.1.13). OAT is a mitochondrial enzyme containing pyridoxal-5'-phosphate as a cofactor, which catalyzes the conversion of L-ornithine to L-glutamate gamma-semialdehyde using 2-oxoglutarate as a terminal amino group acceptor. It has been described in humans, animals, insects, plants and microorganisms. Based on the crystal structure of human OAT, both substrate binding and reaction mechanism of the enzyme are well understood. OAT shows a large structural and mechanistic similarity to other enzymes from the subgroup III of aminotransferases, which transfer an amino group from a carbon atom that does not carry a carboxyl function. In plants, the enzyme has been implicated in proline biosynthesis and accumulation (via pyrroline-5-carboxylate), which represents a way to regulate cellular osmolarity in response to osmotic stress. However, the exact metabolic pathway involving OAT remains a subject of controversy.

Recombinant proteins and peptides as tools for studying IgE reactivity with low-molecular-weight glutenin subunits in some wheat allergies.

Two genes of wheat low-molecular-weight glutenin subunits (LMW-GS), B16 and P73, were cloned and expressed in E. coli. They were homologous to proteins encoded respectively at Glu-B3 and Glu-D3 loci. The N-terminal and C-terminal halves of B16 (NB16 and B16C) and the two chimeras combining the halves of the two genes (B16-P73 and P73- B16) were also expressed. All these constructs were compared for their reactivity with IgE from 24 patients suffering from different forms of wheat allergies. The results confirmed that LMW-GSs bound IgE in all adult allergies tested. Strong differences in reactivity between all the constructs were observed. They were disease-dependent. In wheat-dependent exercise-induced anaphylaxis (WDEIA), the reactivity of the constructs depended partly on common epitopes with omega-5 gliadins but also on differences in molecule conformation. The presence of NB16 in the constructs greatly influenced their IgE reactivity.