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

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

• MeSH
• automatizace MeSH
• beta-glukosidasa chemie   MeSH
• katalýza MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• rostlinné proteiny chemie   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

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 have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM-1cm-2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

• MeSH
• biosenzitivní techniky metody   MeSH
• cytokininy analýza   metabolismus   MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• isopentenyladenosin analogy a deriváty   analýza   metabolismus   MeSH
• kukuřice setá enzymologie   MeSH
• limita detekce MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie 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

The maize β-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-D-glucopyranoside versus the trans-zeatin-O-β-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.

• MeSH
• aminokyseliny metabolismus   MeSH
• beta-glukosidasa chemie   genetika   MeSH
• cytokininy metabolismus   MeSH
• glukosidy metabolismus   MeSH
• hydrolýza MeSH
• isomerie MeSH
• kukuřice setá chemie   enzymologie   genetika   MeSH
• molekulární konformace MeSH
• mutace MeSH
• mutageneze cílená metody   MeSH
• rostlinné geny MeSH
• rostlinné proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zeatin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

• MeSH
• beta-glukosidasa genetika   metabolismus   MeSH
• chloroplasty účinky léků   metabolismus   MeSH
• cytokininy metabolismus   MeSH
• fazol genetika   MeSH
• geneticky modifikované rostliny MeSH
• glukosidy metabolismus   MeSH
• hybridizace genetická MeSH
• kukuřice setá enzymologie   genetika   MeSH
• semenáček cytologie   účinky léků   růst a vývoj   MeSH
• tabák cytologie   účinky léků   genetika   metabolismus   MeSH
• vakuoly účinky léků   genetika   metabolismus   MeSH
• zeatin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.

• MeSH
• 2D gelová elektroforéza MeSH
• antioxidancia metabolismus   MeSH
• dehydratace MeSH
• fyziologická adaptace MeSH
• genotyp MeSH
• glutathionreduktasa metabolismus   MeSH
• katalasa metabolismus   MeSH
• kukuřice setá enzymologie   genetika   fyziologie   MeSH
• období sucha MeSH
• proteomika MeSH
• průduchy rostlin fyziologie   MeSH
• superoxiddismutasa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH