In recent years, substantial progress has been made in exploring auxin conjugation and metabolism, primarily aiming at indole-3-acetic acid (IAA). However, the metabolic regulation of another key auxin, phenylacetic acid (PAA), remains largely uncharacterized. Here, we provide a comprehensive exploration of PAA metabolism in land plants. Through LC-MS screening across multiple plant species and their organs, we identified four previously unreported endogenous PAA metabolites: phenylacetyl-leucine (PAA-Leu), phenylacetyl-phenylalanine (PAA-Phe), phenylacetyl-valine (PAA-Val), and phenylacetyl-glucose (PAA-glc). Enzyme assays, genetic evidence, crystal structures, and docking studies demonstrate that PAA and IAA share core metabolic machinery, revealing a complex regulatory network that maintains auxin homeostasis. Furthermore, our study of PAA conjugation with amino acids and glucose suggests limited compensatory mechanisms within known conjugation pathways, pointing to the existence of alternative metabolic routes in land plants. These insights advance our knowledge of auxin-specific metabolic networks and highlight the unique complexity within plant hormone regulation.

• Klíčová slova
• Gretchen Hagen 3, HPLC-MS/MS, auxin, conjugation, glucosyl ester, indole-3-acetic acid, metabolism, phenylacetic acid, plant,
• Publikační typ
• časopisecké články MeSH

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The most active compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (identified as 82) activated a cytokinin primary response gene, ARR5:GUS, and a cytokinin sensor, TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the up-regulation of cytokinin signalling. Overall, this study identifies highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

• Klíčová slova
• Agriculture, Arabidopsis, CKX inhibitor, biostimulant, biotechnology, cytokinin, cytokinin oxidase/dehydrogenase, diphenylurea, nutrient use efficiency, oilseed rape,
• MeSH
• Arabidopsis * účinky léků   genetika   MeSH
• Brassica napus genetika   účinky léků   MeSH
• cytokininy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• kukuřice setá účinky léků   genetika   růst a vývoj   MeSH
• oxidoreduktasy * metabolismus   genetika   MeSH
• rostlinné proteiny metabolismus   genetika   chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zemědělství MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy * MeSH
• rostlinné proteiny MeSH

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

• Klíčová slova
• Physcomitrium patens, Zea mays, N-acetyl transferase, X-ray crystallography, acetylation, coenzyme A, lysine, ornithine, polyamine, spermine,
• MeSH
• acetylace MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• katalýza MeSH
• kukuřice setá metabolismus   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• ornithin metabolismus   MeSH
• polyaminy * metabolismus   MeSH
• savci metabolismus   MeSH
• spermidin * MeSH
• spermin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetyltransferasy MeSH
• lysin MeSH
• ornithin MeSH
• polyaminy * MeSH
• spermidin * MeSH
• spermin MeSH

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10-8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

• Klíčová slova
• Agriculture, CKX inhibitor, biotechnology, crystal structure, cytokinin, cytokinin oxidase/dehydrogenase, diphenylurea, plant tissue culture, stress, yield,
• MeSH
• Arabidopsis * MeSH
• biotechnologie MeSH
• cytokininy MeSH
• oxidoreduktasy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• oxidoreduktasy * MeSH

Methionine deprivation induces growth arrest and death of cancer cells. To eliminate l-methionine we produced, purified, and characterized the recombinant pyridoxal 5'-phosphate (PLP)-dependent l-methionine γ-lyase (MGL)- BL929 from the cheese-ripening Brevibacterium aurantiacum Transformation of an Escherichia coli strain with the gene BL929 from B. aurantiacum optimized for E. coli expression led to production of the MGL-BL929. Elimination of l-methionine and cytotoxicity in vitro were assessed, and methylation-sensitive epigenetics was explored for changes resulting from exposure of cancer cells to the enzyme. A bioreactor was built by encapsulation of the protein in human erythrocytes to achieve sustained elimination of l-methionine in extracellular fluids. Catalysis was limited to α,γ-elimination of l-methionine and l-homocysteine. The enzyme had no activity on other sulfur-containing amino acids. Enzyme activity decreased in presence of serum albumin or plasma resulting from reduction of PLP availability. Elimination of l-methionine induced cytotoxicity on a vast panel of human cancer cell lines and spared normal cells. Exposure of colorectal carcinoma cells to the MGL-BL929 reduced methyl-CpG levels of hypermethylated gene promoters including that of CDKN2A, whose mRNA expression was increased, together with a decrease in global histone H3 dimethyl lysine 9. The MGL-erythrocyte bioreactor durably preserves enzyme activity in vitro and strongly eliminates l-methionine from medium.

• MeSH
• bioreaktory MeSH
• Brevibacterium enzymologie   MeSH
• dospělí MeSH
• erytrocyty účinky léků   metabolismus   MeSH
• lidé MeSH
• lyasy štěpící vazby C-S farmakologie   MeSH
• methionin metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• rekombinantní proteiny farmakologie   MeSH
• tobolky MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• L-methionine gamma-lyase MeSH Prohlížeč
• lyasy štěpící vazby C-S MeSH
• methionin MeSH
• rekombinantní proteiny MeSH
• tobolky 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.

• Klíčová slova
• Physcomitrella patens, ALDH21, ALDH5, X-ray crystallography, aldehyde dehydrogenase, site-directed mutagenesis, structure-function, succinic semialdehyde, γ-aminobutyric acid,
• 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
• Názvy látek
• GABA MeSH
• kyselina jantarová MeSH
• succinic semialdehyde MeSH Prohlížeč
• sukcinátsemialdehyddehydrogenasa MeSH

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.

• Klíčová slova
• Crystal structure, Cytokinin, Cytokinin oxidase/dehydrogenase, Molecular docking, Organic synthesis, Thidiazuron,
• MeSH
• cytokininy metabolismus   MeSH
• fenylmočovinové sloučeniny chemie   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• oxidoreduktasy antagonisté a inhibitory   MeSH
• thiadiazoly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• fenylmočovinové sloučeniny MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy MeSH
• thiadiazoly MeSH
• thidiazuron MeSH Prohlížeč

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

• Klíčová slova
• allergen, cysteine protease, immunotherapy, protein processing, ragweed, structure-function,
• MeSH
• alergeny chemie   imunologie   MeSH
• antigeny rostlinné imunologie   MeSH
• cysteinové proteasy chemie   imunologie   MeSH
• katalytická doména MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• posttranslační úpravy proteinů MeSH
• prekurzory enzymů chemie   imunologie   MeSH
• proteolýza MeSH
• rostlinné extrakty imunologie   MeSH
• rostlinné proteiny chemie   imunologie   MeSH
• sekvence aminokyselin MeSH
• sezónní alergická rýma imunologie   prevence a kontrola   MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alergeny MeSH
• antigeny rostlinné MeSH
• cysteinové proteasy MeSH
• prekurzory enzymů MeSH
• ragweed pollen MeSH Prohlížeč
• rostlinné extrakty MeSH
• rostlinné proteiny MeSH

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP(+) converting trans-zeatin (tZ) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of tZ side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of tZ and cis-zeatin (cZ). While tZ is preferentially glucosylated at the N9 position, cZ forms mainly O-glucosides. Since O-glucosides, in contrast to N9-glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O-glycosylate zeatin in the cis-position might be a reason why cZ derivatives are over-accumulated in different maize tissues and organs.

• Klíčová slova
• Anticytokinin, Cytokinin, Kernels, Metabolism, Zea mays L., cis-zeatin, trans-zeatin,
• MeSH
• cytokininy analýza   izolace a purifikace   metabolismus   MeSH
• glykosylace MeSH
• glykosyltransferasy metabolismus   MeSH
• kukuřice setá růst a vývoj   metabolismus   MeSH
• opylení MeSH
• oxidoreduktasy metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin analýza   izolace a purifikace   metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• semena rostlinná růst a vývoj   metabolismus   MeSH
• semenáček růst a vývoj   metabolismus   MeSH
• terpeny analýza   izolace a purifikace   metabolismus   MeSH
• zeatin analýza   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• glykosyltransferasy MeSH
• oxidoreduktasy MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny MeSH
• terpeny MeSH
• zeatin 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.

• Klíčová slova
• crystal structure, cytokinin oxidase/dehydrogenase, flavoprotein, maize, plant hormone, site-directed mutagenesis,
• 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
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• flavinadenindinukleotid MeSH
• oxidoreduktasy MeSH