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Site‐directed mutagenesis of bovine deoxyhypusine hydroxylase at the hinge loop area
Author(s) -
Murtaugh Megan L,
Wang Yinglu,
Smith Joshua L,
Soustek Meghan S,
Appidi Vidya,
Huang JenqKuen,
Wen Lisa
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.625.3
Subject(s) - mutant , site directed mutagenesis , mutagenesis , complementary dna , biology , biochemistry , amino acid , enzyme , directed evolution , chaperone (clinical) , microbiology and biotechnology , dna , gene , medicine , pathology
Deoxyhypusine hydroxylase (DOHH) is the second of the two enzymes that catalyze the maturation 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. We have recently cloned bovine DOHH cDNA. The deduced amino acid sequence shared 88% identity with human DOHH. The dual function of human DOHH has been reported: the Fe containing holoenzyme exhibited hydroxylase activity while the apoenzyme showed molecular chaperone activity (Kang et al ., The FASEB Journal . 2007;21:lb122). The structure transition between the holoenzyme and the apoenzyme is believed to take place around the variable hinge loop area. In this work, we prepare three amino acid substitution mutants at the loop area: P143S (Pro at the position 143 to Ser), D148A (Asp at 148 to Ala), and E154V (Glu at 154 to Val) of the bovine DOHH. The mutant DNA has been created by site‐directed mutagenesis using PCR primed with mutagenic primers followed by overlap extension. The mutant DNA is subcloned into pGEX‐2T, overexpressed, and characterized. We will compare the hydroxylase and chaperone activities of the mutant proteins to the wild‐type DOHH. Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.