Western Illinois University1, Chemistry, Macomb, IL 61455 Western Illinois Universty2, Chemistry, Macomb, IL 61455
Deoxyhypusine hydroxylase (DOHH) is the second of the two enzymes that catalyze the maturation/hypusination of eukaryotic initiation factor 5A (eIF5A). It has been well established that hypusinated eIF5A is essential for eukaryotic cell proliferation and survival and that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy. A detailed structure-function study of the enzyme will help in designing specific inhibitors to better control hyperproliferative diseases.
We have recently cloned bovine DOHH cDNA (Huang et al., Protein Expr. Purif. 54, 126-133, 2007). The deduced amino acid sequence shared 88% identity with human DOHH. The recombinant human DOHH has been shown to possess dual function as an enzyme for eIF5A maturation and a molecular chaperone: the iron containing holoenzyme exhibited hydroxylase activity while the apoenzyme exhibited molecular chaperone activity (Kang et al., The FASEB Journal. 2007;21:lb122 ). The structure transition between the holoenzyme and the apoenzyme was believed to take place around the variable hinge loop area. In this research, we will create amino acid substitution mutants at this loop area. Here, we report the preparation of single amino acid substitution mutants P143S (proline at the position 143 to serine), D148A (asparagine at the position 148 to alanine), and E154V (glutamate at the position 154 to valine) of the bovine DOHH. The mutant DNA has been prepared by site-directed mutagenesis using polymerase chain reaction (PCR) primed with mutagenic oligonucleotides followed by overlap extension. The mutant DNA will be subcloned into an expression vector, pGEX-2T. The recombinant protein will be overexpressed and characterized. We will compare the hydroxylase and chaperone activities of the mutant proteins to the wild-type DOHH.
Supported in part by the University Research Council, Western Illinois University.
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