Deoxyhypusine hydroxylase: a novel HEAT‐repeat‐containing metalloenzyme

Abstract

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the formation of the hypusine residue, uniquely found in eukaryotic translation initiation factor 5A (eIF5A). Although we have studied properties of DOHH in cells and partially purified from rat testis, its identity has been elusive. Recently we cloned the DOHH gene by screening a S. cerevisiae GST-ORF library (constructed by Dr. Eric M. Phizicky, University of Rochester), identified the human DOHH gene and expressed catalytically active recombinant enzymes. A homology-based model indicates that the protein has 8 distinct HEAT-repeat motifs arranged in tandem to form a superhelical solonoid structure. The essentially alpha-helical nature of the protein is confirmed by its circular dichroism UV spectrum (80% alpha-helix). DOHH contains two potential metal binding sites, each made up of a strictly conserved His-Glu pair. The purified DOHH displayed a distinctive blue color and indeed Fe was identified as the DOHH-bound metal. The role of the His-Glu motif was investigated by site-directed mutagenesis. Alanine substitution of H56, H89, E90, H207, H240 and E241 caused a marked reduction in Fe content and alanine substitution of any residues of the HisGlu motifs abolished DOHH activity, indicating their critical role in metal coordination and catalysis. This research was supported by the Intramural Research Program of the NIH (NIDCR, NCBI/NLM)