High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

Kopečný, David; Pethe, Claude; Šebela, Marek; Houba-Hérin, Nicole; Madzak, Catherine; Majira, Amel; Laloue, Michel

doi:10.1016/j.biochi.2005.04.006

Cited by 42 publications

(35 citation statements)

References 34 publications

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“…Resting oxidized ZmCKO1 provides an absorption spectrum with two distinct maxima at 360 and 445 nm in the visible region. 10 The addition of HA-1 and HA-8 in a 50:1 excess resulted in complete bleaching of these absorption bands within 10 s. No bleaching was observed with HA-10 or when the compounds were mixed with free FAD (Fig. 2a), showing (particularly in the latter case) that bleaching occurs only as a result of enzymatic reaction.…”

Section: Kinetic and Spectral Analyses Of Zmcko1 Inhibitionmentioning

confidence: 83%

“…Similarly, the addition of HA-8 or HA-1 abolished fluorescence properties of the enzyme. The characteristic fluorescence emission (λ max = 525 nm; excitation at 450 nm) and excitation (λ max = 360 and 450 nm; emission at 525 nm) spectra, which were previously reported, 10 could not be measured using the inactivated enzyme even after exhaustive dialysis. The reaction of ZmCKO1 with HA-8 or HA-1 also resulted in bleaching of the electron acceptor 2,6-dichlorophenol indophenol (DCPIP), indicating FAD reoxidation during catalysis.…”

Section: Kinetic and Spectral Analyses Of Zmcko1 Inhibitionmentioning

confidence: 99%

“…Our previous results using mass spectrometry showed the presence of about 84 hexose units attributed to five N-glycosylation sites. 10 The electron density map Fig. 3.…”

Section: Raman Spectroscopy Of Inactivated Zmcko1mentioning

confidence: 99%

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Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

Kopečný

Šebela

Briozzo

et al. 2008

Journal of Molecular Biology

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Section: Kinetic and Spectral Analyses Of Zmcko1 Inhibitionmentioning

confidence: 83%

Section: Kinetic and Spectral Analyses Of Zmcko1 Inhibitionmentioning

confidence: 99%

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Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

Kopečný

Šebela

Briozzo

et al. 2008

Journal of Molecular Biology

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“…These separation processes usually consist of salting-out, detergent extraction, heat treatment, and chromatography [8][9][10][11][12][13][14][15][16]. The large-scale preparation of flavoenzymes calls for more universal and efficient protocols.…”

Section: Discussionmentioning

confidence: 99%

“…was purified through ammonium sulfate precipitation, ion-exchange chromatography on diethylaminoethyl-cellulose column, and hydrophobic chromatography on a Butyl-Toyopearl column [8]. Cytokinin oxidase/dehydrogenase from Yarrowia lipolytica was purified through ammonium sulfate precipitation, ion-exchange chromatography on a Resource Q column, and chromatography on a Sephacryl S-200 column [9]. Alcohol oxidase from Aspergillus terreus was purified through ammonium sulfate precipitation and chromatography on a diethylaminoethyl column [10].…”

Section: Introductionmentioning

confidence: 99%

Preparation and characterization of affinity sorbents based on isoalloxazine‐like ligands for separation of flavoenzymes

Xin

Yang

Xiao

et al. 2011

J of Separation Science

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Affinity ligands for flavoenzymes were synthesized based on the natural structure of flavo-coenzymes. Two typical flavoenzymes, cholesterol oxidase from Brevibacterium sp. and xanthine oxidase from bovine milk, were employed as standard enzymes. Fluorescent probes were synthesized from eight isoalloxazine-like chemicals and 5-aminofluorescein. Probe-enzyme interactions were analyzed via fluorescence spectra. Chemicals with high binding abilities to flavoenzymes were coupled with Sepharose through spacers composed of epichlorohydrin, ethylenediamine, 1,3-diaminopropane, 2-hydroxy-1,3-diaminopropane, and 1,4-diaminobutane, and subjected to adsorption analysis with flavoenzymes. The results indicated that ligands synthesized from 2,4-dioxohexahydropyrimidine-5-carboxylic acid, cytosine, 7-chloroalloxazine, and 8-chloroalloxazine had high binding abilities to the flavoenzymes. The affinity sorbent based on these ligands revealed a high theoretical maximum adsorption (Q(max)). Protein and bioactivity recoveries were tested after one step of affinity binding via chromatographic analysis on small columns. Results showed that ligands linked with sorbents through long hydrophilic spacers had higher activity recoveries.

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Identification of N‐glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics

et al. 2009

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An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis.

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High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

Cited by 42 publications

References 34 publications

Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

Preparation and characterization of affinity sorbents based on isoalloxazine‐like ligands for separation of flavoenzymes

Identification of N‐glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics

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