Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD+-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family.
Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes, which detoxify aldehydes produced in various metabolic pathways to the corresponding carboxylic acids. Among the 19 human ALDHs, the cytosolic ALDH9A1 has so far never been fully enzymatically characterized and its structure is still unknown. Here, we report complete molecular and kinetic properties of human ALDH9A1 as well as three crystal forms at 2.3, 2.9, and 2.5 Å resolution. We show that ALDH9A1 exhibits wide substrate specificity to aminoaldehydes, aliphatic and aromatic aldehydes with a clear preference for γ-trimethylaminobutyraldehyde (TMABAL). The structure of ALDH9A1 reveals that the enzyme assembles as a tetramer. Each ALDH monomer displays a typical ALDHs fold composed of an oligomerization domain, a coenzyme domain, a catalytic domain, and an inter-domain linker highly conserved in amino-acid sequence and folding. Nonetheless, structural comparison reveals a position and a fold of the inter-domain linker of ALDH9A1 never observed in any other ALDH so far. This unique difference is not compatible with the presence of a bound substrate and a large conformational rearrangement of the linker up to 30 Å has to occur to allow the access of the substrate channel. Moreover, the αβE region consisting of an α-helix and a β-strand of the coenzyme domain at the dimer interface are disordered, likely due to the loss of interactions with the inter-domain linker, which leads to incomplete β-nicotinamide adenine dinucleotide (NAD+) binding pocket.
- MeSH
- aldehyddehydrogenasa antagonisté a inhibitory chemie genetika ultrastruktura MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- konformace proteinů * MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- NAD genetika MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin genetika MeSH
- substrátová specifita genetika MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MAIN CONCLUSION: Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.
- MeSH
- alkyltransferasy a aryltransferasy genetika metabolismus MeSH
- Arabidopsis genetika metabolismus MeSH
- cytokininy biosyntéza MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- listy rostlin genetika metabolismus MeSH
- Populus genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
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
Lower plant species including some green algae, non-vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP+ -dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ-aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD+ is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP+ binding induces a conformational change of the loop carrying Arg-228, which seals the NADP+ in the coenzyme cavity via its 2'-phosphate and α-phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg-121 and Arg-457, and a hydrogen bond with Tyr-296. While both arginine residues are pre-formed for substrate/product binding, Tyr-296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5.
- MeSH
- Bryophyta enzymologie genetika MeSH
- fylogeneze MeSH
- GABA analogy a deriváty metabolismus MeSH
- kapradiny enzymologie genetika MeSH
- konformace proteinů MeSH
- kyselina jantarová metabolismus MeSH
- rostlinné geny genetika fyziologie MeSH
- substrátová specifita MeSH
- sukcinátsemialdehyddehydrogenasa genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.
- MeSH
- cytokininy metabolismus MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kukuřice setá enzymologie MeSH
- mutageneze cílená MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
KEY MESSAGE: Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.
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
We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.
- MeSH
- biokatalýza * účinky léků MeSH
- cytokininy chemie metabolismus MeSH
- dusík farmakologie MeSH
- fenotyp MeSH
- fylogeneze MeSH
- genový knockout MeSH
- hydrolýza účinky léků MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kukuřice setá účinky léků enzymologie genetika MeSH
- mechy účinky léků enzymologie genetika růst a vývoj MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie metabolismus MeSH
- N-glykosylhydrolasy chemie genetika metabolismus MeSH
- pyrimidiny chemie metabolismus MeSH
- ribonukleosidy chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- substrátová specifita účinky léků MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aldehydoxidoreduktasy chemie genetika metabolismus MeSH
- aldehydy chemie MeSH
- chemické modely MeSH
- fylogeneze MeSH
- fyziologie rostlin MeSH
- kinetika MeSH
- krystalografie rentgenová metody MeSH
- kukuřice setá enzymologie MeSH
- mutageneze cílená MeSH
- NAD chemie MeSH
- polyethylenglykoly chemie MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u rostlin * MeSH
- rostliny enzymologie MeSH
- semena rostlinná metabolismus MeSH
- Solanum lycopersicum enzymologie MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
