Structure-function
study on enzymes linked to cytokinin metabolism - aldehyde dehydrogenases and
nucleoside N-ribohydrolases
Radka Končitíková1,2, Martina Kopečná1, Armelle Vigouroux3, Solange Moréra3, Marek Šebela1, David Kopečný1,*
1Department
of Protein Biochemistry and Proteomics, Centre of the Region Hana, Faculty of
Science, Palacký University, Olomouc, Czech Republic
2Department of Biochemistry, Faculty of Science, Palacký University,
Olomouc, Czech Republic
3Laboratoire d'Enzymologie et Biochimie Structurales, CNRS,
Gif-sur-Yvette, France
Plant hormones cytokinins are irreversibly inactivated by cytokinin oxidase/dehydrogenase (CKO). Adenosine/adenine moiety and corresponding aldehyde are produced in this metabolic pathway. We focused on two enzyme groups possibly involved in metabolism of these products. They include aldehyde dehydrogenases (ALDHs) oxidizing aldehydes to corresponding acids and nucleoside N-ribohydrolases (NRHs) catalyzing conversion of adenosine to adenine. While nucleoside N-ribohydrolases (E.C. 3.2.2.-) belong to the family of hydrolases catalyzing the cleavage of the N-glycosidic bond in purine and pyrimidine nucleosides, aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases (EC 1.2.1.-). ALDHs are considered as general detoxifying enzymes, which eliminate biogenic and xenobiotic aldehydes to the corresponding carboxylic acids. Up to date, 13 ALDH families have been described in plants but only a small number of the enzymes have been functionally characterized despite the existence of a large number of coding genes. We analyzed ALDH2 and ALDH7 families from maize (Zea mays). The ALDH2 family expanded significantly during evolution of terrestrial plants and the number of family members varies substantially between species. Maize ALDH2 (ZmALDH2) includes six genes coding for mitochondrial and cytosolic enzymes while ALDH7 family includes only one gene. Some ALDH2 family members were originally identified as genes restoring fertility in plants (called RF2), which have the ability to suppress the male-sterile phenotype and restore the production of pollen to plants carrying the deleterious mitochondrial genome. Two genes RF2C and RF2F coding for two ZmALDH2 enzymes and the gene coding for ZmALDH7 were cloned and expressed in E. coli. The obtained recombinant proteins were thoroughly characterized and their identity was verified by MALDI-TOF peptide mass fingerprinting. All studied ALDHs utilized NAD+ but not NADP+ as a coenzyme. They were crystallized and their crystal structures were solved. They represent the first structures of plant ALDH2 and ALDH7 family members. Gene expression analysis using TaqMan probes revealed RF2C as a predominant ALDH2 gene expressed in all plant tissues while other ALDH2 genes are less abundant and specific to some developmental stages.
This work was supported by grant P501/11/1591 from the Czech Science Foundation, LO1204 from the Ministry of Education, Youth and Sports of the Czech Republic and IGA grant from Faculty of Sciences, Palacký University.