Identification and characterization of a novel deoxyhypusine synthase in Leishmania donovani.
Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.
Chawla
Bhavna
B
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Jhingran
Anupam
A
Singh
Sushma
S
Tyagi
Nidhi
N
Park
Myung Hee
MH
Srinivasan
N
N
Roberts
Sigrid C
SC
Madhubala
Rentala
R
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D004926:Escherichia coli; D017353:Gene Deletion; D016678:Genome / Q000235:genetics; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010271:Parasites / Q000201:enzymology / Q000235:genetics; D010802:Phylogeny; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D015800:Protozoan Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase