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

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 degrees C for 30 min incubation). The following pH optimum and K(m) values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (K(m)=0.5 microM), pH 7.6 for isopentenyl adenosine (K(m)=1.9 microM), pH 7.9 for zeatin (K(m)=1.5 microM) and pH 7.3 for zeatin riboside (K(m)=2.0 microM). 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).

Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs.

Cytokinin oxidases (CKOs) play a major role in the regulation of hormone levels in plants by irreversibly degrading cytokinins. Two new cDNAs from maize (CKO2 and CKO3) were cloned and CKO activity of a recombinant CKO3 enzyme was demonstrated. CKO2 and CKO3 encode flavoproteins with 93% identity among each other compared with 45% identity with CKO1. The respective genes were mapped to BIN 3.05/06 and BIN 8.06 which belong to duplicated regions of the maize genome. For a better understanding of the role of CKO2 and CKO3 in maize development, their expression profiles were analysed in different organs and during kernel development via semi-quantitative RT-PCR. Different spatial and temporal expression patterns were observed for the two genes, as well as for CKO1 and two additional genes CKO4 and CKO5. CKO2 to CKO5 genes were mainly expressed in vegetative tissues, with unique expression patterns. CKO1 was most strongly expressed in the kernel. All five genes were expressed at early stages of kernel development, a period when a peak in cytokinin levels and a high cell division rate in the endosperm have been described. However, each gene had its own expression profile with a major difference concerning the onset of expression.

Purification, crystallization and preliminary X-ray diffraction study of a recombinant cytokinin oxidase from Zea mays.

Cytokinins are hormones that are involved in plant growth and development. They are irreversibly degraded by cytokinin oxidases/dehydrogenases, flavoenzymes which contain a covalently bound flavine adenine dinucleotide (FAD) cofactor. Cytokinin oxidase from Zea mays (ZmCKO1) was overexpressed in the yeast Yarrowia lipolytica, purified (molecular weight 69 kDa) and crystallized using the hanging-drop method. Crystals belong to the monoclinic space group C2, with unit-cell parameters a = 250.6, b = 50.6, c = 51.5 A, beta = 94.1 degrees. A complete data set has been collected at 100 K to 1.95 A resolution on an X-ray synchrotron source.