Plant cytosolic aldehyde dehydrogenases from family 2 (ALDH2s, EC 1.2.1.3) are non-specific enzymes and participate for example in the metabolism of acetaldehyde or biosynthesis of phenylpropanoids. Plant aminoaldehyde dehydrogenases (AMADHs, ALDH10 family, EC 1.2.1.19) are broadly specific and play an important role in polyamine degradation or production of osmoprotectants. We have tested imidazole and pyrazole carbaldehydes and their alkyl-, allyl-, benzyl-, phenyl-, pyrimidinyl- or thienyl-derivatives as possible substrates of plant ALDH2 and ALDH10 enzymes. Imidazole represents a building block of histidine, histamine as well as certain alkaloids. It also appears in synthetic pharmaceuticals such as imidazole antifungals. Biological compounds containing pyrazole are rare (e.g. pyrazole-1-alanine and pyrazofurin antibiotics) but the ring is often found as a constituent of many synthetic drugs and pesticides. The aim was to evaluate whether aldehyde compounds based on azole heterocycles are oxidized by the enzymes, which would further support their expected role as detoxifying aldehyde scavengers. The analyzed imidazole and pyrazole carbaldehydes were only slowly converted by ALDH10s but well oxidized by cytosolic maize ALDH2 isoforms (particularly by ALDH2C1). In the latter case, the respective Km values were in the range of 10-2000 μmol l-1; the kcat values appeared mostly between 0.1 and 1.0 s-1. The carbaldehyde group at the position 4 of imidazole was oxidized faster than that at the position 2. Such a difference was not observed for pyrazole carbaldehydes. Aldehydes with an aromatic substituent on their heterocyclic ring were oxidized faster than those with an aliphatic substituent. The most efficient of the tested substrates were comparable to benzaldehyde and p-anisaldehyde known as the best aromatic aldehyde substrates of plant cytosolic ALDH2s in vitro.
- MeSH
- aldehyddehydrogenasa metabolismus MeSH
- aldehydy chemie metabolismus MeSH
- hrách setý enzymologie MeSH
- imidazoly chemie metabolismus MeSH
- kukuřice setá enzymologie MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- pyrazoly chemie metabolismus MeSH
- Solanum lycopersicum enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- aldehyddehydrogenasa chemie metabolismus MeSH
- aldehydy chemie metabolismus MeSH
- hrách setý chemie enzymologie MeSH
- kinetika MeSH
- molekulární struktura MeSH
- propylaminy chemie metabolismus MeSH
- rostlinné proteiny chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aldehyde dehydrogenases (ALDHs) are responsible for oxidation of biogenic aldehyde intermediates as well as for cell detoxification of aldehydes generated during lipid peroxidation. So far, 13 ALDH families have been described in plants. In the present study, we provide a detailed biochemical characterization of plant ALDH2 and ALDH7 families by analysing maize and pea ALDH7 (ZmALDH7 and PsALDH7) and four maize cytosolic ALDH(cALDH)2 isoforms RF2C, RF2D, RF2E and RF2F [the first maize ALDH2 was discovered as a fertility restorer (RF2A)]. We report the crystal structures of ZmALDH7, RF2C and RF2F at high resolution. The ZmALDH7 structure shows that the three conserved residues Glu(120), Arg(300) and Thr(302) in the ALDH7 family are located in the substrate-binding site and are specific to this family. Our kinetic analysis demonstrates that α-aminoadipic semialdehyde, a lysine catabolism intermediate, is the preferred substrate for plant ALDH7. In contrast, aromatic aldehydes including benzaldehyde, anisaldehyde, cinnamaldehyde, coniferaldehyde and sinapaldehyde are the best substrates for cALDH2. In line with these results, the crystal structures of RF2C and RF2F reveal that their substrate-binding sites are similar and are formed by an aromatic cluster mainly composed of phenylalanine residues and several nonpolar residues. Gene expression studies indicate that the RF2C gene, which is strongly expressed in all organs, appears essential, suggesting that the crucial role of the enzyme would certainly be linked to the cell wall formation using aldehydes from phenylpropanoid pathway as substrates. Finally, plant ALDH7 may significantly contribute to osmoprotection because it oxidizes several aminoaldehydes leading to products known as osmolytes.
- MeSH
- aldehyddehydrogenasa chemie genetika metabolismus MeSH
- fylogeneze MeSH
- hrách setý enzymologie genetika MeSH
- izoenzymy chemie genetika metabolismus MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kukuřice setá enzymologie genetika MeSH
- modely genetické MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- NAD metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- rostliny enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- stanovení celkové genové exprese MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S-nitrosylation of protein cysteine thiol groups has recently emerged as a widespread and important reversible post-translational protein modification, involved in redox signalling pathways of nitric oxide and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR), member of class III alcohol dehydrogenase family (EC 1.1.1.1), is considered the key enzyme in the catabolism of major low molecular S-nitrosothiol, S-nitrosoglutathione, and hence to control the level of protein S-nitrosylation. Changes of GSNOR activity after exposure to different abiotic stress conditions, including low and high temperature, continuous dark and de-etiolation, and mechanical injury, were investigated in important agricultural plants. Significantly higher GSNOR activity was found under normal conditions in leaves of Cucumis spp. genotype sensitive to biotrophic pathogen Golovinomyces cichoracearum. GSNOR activity was generally increased in all studied plants by all types of stress conditions. Strong down-regulation of GSNOR was observed in hypocotyls of etiolated pea plants, which did not recover to values of green plants even 168 h after the transfer of etiolated plants to normal light regime. These results point to important role of GSNOR during normal plant development and in plant responses to several types of abiotic stress conditions.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- Ascomycota patogenita MeSH
- Cucumis melo enzymologie genetika mikrobiologie MeSH
- Cucumis sativus enzymologie genetika mikrobiologie MeSH
- fyziologický stres * MeSH
- hrách setý enzymologie mikrobiologie MeSH
- hypokotyl enzymologie MeSH
- mechanický stres MeSH
- nízká teplota MeSH
- reakce na tepelný šok MeSH
- světlo MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aldehydoxidoreduktasy chemie MeSH
- aldehydy chemie MeSH
- aminokyselinové motivy MeSH
- heterocyklické sloučeniny chemie MeSH
- hrách setý enzymologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- rekombinantní proteiny chemie MeSH
- rostlinné proteiny chemie MeSH
- Solanum lycopersicum enzymologie MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aldehydoxidoreduktasy chemie genetika izolace a purifikace metabolismus MeSH
- aldehydy metabolismus MeSH
- aminokyseliny aromatické metabolismus MeSH
- aminokyseliny dikarboxylové metabolismus MeSH
- betain analogy a deriváty metabolismus MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- hrách setý enzymologie MeSH
- izoenzymy metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie izolace a purifikace metabolismus MeSH
- propylaminy metabolismus MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- rostlinné proteiny chemie genetika izolace a purifikace metabolismus MeSH
- sekundární struktura proteinů MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biokatalýza MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hrách setý enzymologie MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- polyaminy farmakologie MeSH
- rekombinantní proteiny antagonisté a inhibitory chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- substrátová specifita MeSH
- transaminasa ornithin-oxokyselin antagonisté a inhibitory chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aldehyddehydrogenasa chemie izolace a purifikace metabolismus MeSH
- DNA primery MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- financování organizované MeSH
- hrách setý enzymologie MeSH
- klonování DNA MeSH
- konformace proteinů MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- sekvence nukleotidů MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- western blotting MeSH
