title: A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani.
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.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Bhavna
authors.initials: B
authors.lastname: Chawla
authors.forename: Ravi Ranjan
authors.initials: RR
authors.lastname: Kumar
authors.forename: Nidhi
authors.initials: N
authors.lastname: Tyagi
authors.forename: Gowri
authors.initials: G
authors.lastname: Subramanian
authors.forename: N
authors.initials: N
authors.lastname: Srinivasan
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Park
authors.forename: Rentala
authors.initials: R
authors.lastname: Madhubala
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016054:DNA, Protozoan / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017125:Genes, Protozoan; D006801:Humans; D066298:In Vitro Techniques; D007893:Leishmania donovani / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D008239:Lysine; D006899:Mixed Function Oxygenases; D010448:Peptide Initiation Factors; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C047991:deoxyhypusine hydroxylase