STRUCTURAL CHARACTERIZATION OF TWO MAIZE ALDEHYDE DEHYDROGENASES FROM FAMILY 2

Radka Končitíková^1,2, Martina Kopečná¹, David Kopečný¹, Solange Moréra³, Armelle Vigouroux³, Jan Frömmel^1,2, Marek Šebela¹

¹Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research and
²Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic;
³Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, F-91198 Gif-sur-Yvette Cedex, France

Aldehyde dehydrogenases (ALDHs) constitute a protein superfamily of NAD(P)⁺-dependent enzymes (EC 1.2.1). They have been considered as general detoxifying enzymes, which eliminate biogenic and xenobiotic aldehydes to the corresponding carboxylic acids. Under conditions of oxidative stress, ALDH enzymes act as aldehyde scavengers by metabolizing reactive aldehydes produced in consequence of the oxidative degradation of lipid membranes, also known as lipid peroxidation. 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. In this work, we focused on ALDH gene family 2 in maize (Zea mays). The ALDH2 family expanded significantly during evolution of terrestrial plants and the number of family members varies substantially between species. The maize ALDH2 includes six genes coding for mitochondrial and cytosolic enzymes. Some ALDH2 family members were originally identified as genes restored fertility in plants (called RF2) i.e., gene that 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 ALDHs were cloned and expressed in T7 E. coli cells. The recombinant enzymes were thoroughly characterized and their identity was verified by MALDI-TOF peptide mass fingerprinting. Both ZmALDH2 enzymes utilize NAD⁺ but not NADP⁺ as a coenzyme. They are able to oxidize wide range of aldehydes but prefer aliphatic aldehydes. The crystal structure of ZmALDH2 (ALDH2C1, also RF2C) has been solved up to 2.25 Ǻ resolution with R_cryst and R_free values of 18.2% and 22.5%, respectively, and represents the first structure of plant ALDH2 family member.

This work was supported by grant P501/11/1591 from the Czech Science Foundation and IGA grant PrF_2012_012 from Faculty of Sciences, Palacky University.