Abstract
Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N €-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here 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 α-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. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. 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.
Highlights
Hypusine (NJ-(4-amino-2-hydroxybutyl) lysine) is a unique amino acid present in the eukaryotic initiation factor 5A [1]
L. donovani deoxyhypusine hydroxylase (DOHH) amino acid sequence were performed as described in Materials and Methods
Sequence analysis of the L. donovani DOHH protein showed the presence of eight tandem HEAT repeats
Summary
Hypusine (NJ-(4-amino-2-hydroxybutyl) lysine) is a unique amino acid present in the eukaryotic initiation factor 5A (eIF5A) [1]. Despite conservation of some of the active site amino acid residues between the human and leishmanial DHS, a potent inhibitor of human DHS, N1-guanyl-1, 7-diaminoheptane, had little inhibitory effect on either L. donovani proliferation or recombinant DHS34 This finding suggests a topological difference in the spermidine binding sites between the human and the leishmanial enzymes and opens the possibility that the differences between the two enzymes could be exploited for drug development for visceral leishmaniasis. Metal chelators like CPX and mimosine significantly inhibited the growth of L. donovani and the activity of recombinant DOHH in vitro These inhibitors were much more effective against the L. donovani than the human enzyme. The structural differences between the L. donovani DOHH and the human homolog might account for the differences in the inhibitor binding properties of the parasite compared to those of the human homolog
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