David Kopečný

Publications (5)5.07 Total impact

• Article: Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry.

  Vojtěch Franc, Marek Sebela, Pavel Rehulka, Radka Končitíková, René Lenobel, Catherine Madzak, David Kopečný
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  ABSTRACT: Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.
  Journal of proteomics 05/2012; 75(13):4027-37. · 5.07 Impact Factor
• Article: Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: A structural study

  David Kopečný, Pierre Briozzo, Hana Popelková, Marek Šebela, Radka Končitíková, Lukáš Spíchal, Jaroslav Nisler, Catherine Madzak, Ivo Frébort, Michel Laloue, Nicole Houba-Hérin
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  ABSTRACT: Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme–inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N′-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N6-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N′-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N′-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity.
  Biochimie.
• Article: High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

  David Kopečný, Claude Pethe, Marek Šebela, Nicole Houba-Hérin, Catherine Madzak, Amel Majira, Michel Laloue
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  ABSTRACT: Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50 = 55 °C for 30 min incubation). The following pH optimum and Km values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (Km = 0.5 μM), pH 7.6 for isopentenyl adenosine (Km = 1.9 μM), pH 7.9 for zeatin (Km = 1.5 μM) and pH 7.3 for zeatin riboside (Km = 2.0 μM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors).
  Biochimie.
• Article: Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

  David Kopečný, Marek Šebela, Pierre Briozzo, Lukáš Spíchal, Nicole Houba-Hérin, Vlastimil Mašek, Nathalie Joly, Catherine Madzak, Pavel Anzenbacher, Michel Laloue
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  ABSTRACT: Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N6-(buta-2,3-dienyl)adenine (HA-8) and N6-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.
  Journal of Molecular Biology.
• Source

  Available from: Petr Tarkowski

  Article: Probing cytokinin homeostasis in Arabidopsis thaliana by constitutively overexpressing two forms of the maize cytokinin oxidase/dehydrogenase 1 gene

  David Kopečný, Petr Tarkowski, Amel Majira, Isabelle Bouchez-Mahiout, Fabien Nogué, Michel Laurière, Goran Sandberg, Michel Laloue, Nicole Houba-Hérin
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  ABSTRACT: Engineering transgenic plants with reduced cytokinin (Ck) contents is a way to analyze the role of these hormones in growth and development. Cytokinin oxidase/dehydrogenase (CKO) genes are good candidates to promote Ck deficiency. They code for enzymes degrading Cks and generally belong to multigene families. Plants constitutively expressing naturally occurring CKO genes that code for secreted or vacuolar enzymes have been described. We report on Arabidopsis transgenics constitutively overexpressing the secreted native form or an engineered non-secreted form of the maize CKO1 enzyme. Severity of phenotype symptoms (increased root system, reduced size of aerial parts and defects in seed development) was clearly correlated with the level of enzyme activity. Aerial part was especially affected in plants overexpressing the non-secreted enzyme, even at low activity level. In all strong overexpressers, zeatin-type metabolites were highly depleted compared to isopentenyladenine-type metabolites. AtIPT genes involved in Ck biosynthesis were found to be up-regulated in those transgenics while all AtCKO genes were down-regulated except At5g21482, coding for a putative cytoplasmic enzyme. Cytokinin deficiency in transgenics was not counter-balanced by a higher sensitivity: expression of a cytokinin receptor and type-A response regulators was decreased as well as plant response to benzyladenine.
  Plant Science.