In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). We and others have demonstrated that hypusine modification is essential for the activity of eIF5A and for mammalian cell proliferation. Previously, we reported biochemical evidence for acetylation of eIF5A at Lys47 and its negative regulation by this acetylation. We have also obtained evidence for selective acetylation of hypusinated eIF5A, but not nonhypusinated eIF5A, by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). The site of eIF5A acetylation by SSAT1 was identified as terminal amino group of the hypusine side chain by ion exchange chromatographic separation. The bovine testis eIF5A acetylated by SSAT1 was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. To investigate the physiological function of eIF5A isoforms and the hypusine modification enzymes, we performed their gene targeting in mice using the ES cell lines, RRE174 (Eif5a-1 +/-) and RRM039 (Dhps +/-) which have one allele of each gene disrupted by the gene trap method. The gene-targeted heterozygous agouti mice (Eif5a-1 +/-, or Dhps +/-) appeared to be normal and did not show any growth defects or phenotypes. The heterozygous agouti male and female mice were crossed and the pups born from the heterozygous intercrosses were genotyped. No pups were born with the genotype of Eif5a-1-/- or Dhps-/- indicating that homozygous disruption of either gene is embryonic lethal. To clarify the time point of embryonic lethality, we cultured the embryos at developmental stages (E3.5, E6.5, E7.5 and E8.5) and genotyped them by PCR. The Eif5a-1-/- homozygous embryo was identified on the blastocyst stage (E3.5), but not at later stage, indicating that Eif5a-1-/- embryo is viable up to 3.5 days, but not after 6 days. The same was true for Dhps-/- embryos. These findings demonstrate that eIF5A-1 and DHS play an essential role at the early stage of embryonic development. We investigated the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of -helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A 42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed 40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite. EF-P is a bacterial ortholog of eIF5A and it does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical beta-lysyl-lysine. To obtain biochemical evidence for the hypothesis and to assess the role of this modification in EF-P activity, we have over-expressed EF-P alone, or in combination with YjeA and YjeK in E. coli using a polycistronic vector. Over-expression of EF-P alone, EF-P plus YjeA, and EF-P plus YjeA plus YjeK resulted in unmodified EF-P, alpha-lysyl EF-P and beta-lysyl EF-P, respectively. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native EF-P as well as in recombinant alpha-lysyl or beta-lysyl EF-P proteins. The beta-lysyl-lysine isopeptide was identified in the exhaustive pronase digests of native EF-P and recombinant EF-P isolated from E.coli coexpressing EF-P, YjeA and YjeK, but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for beta-lysylation of EF-P. Endogenous EF-P and the recombinant beta-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis approximately four-fold over the preparations containing unmodified EF-P and/or alpha-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the beta-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.
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