title: Probing the roles of non-homologous insertions in the N-terminal domain of Plasmodium falciparum hydroxymethylpterin pyrophosphokinase-dihydropteroate synthase.
abstract: Plasmodium falciparum bifunctional hydroxymethylpterin pyrophosphokinase-dihydropteroate synthase (pfHPPK-DHPS) is a crucial enzyme in the de novo folate biosynthesis pathway. The crystal structure is not yet available for this enzyme, however, homology model of the enzyme reported previously revealed the presence of parasite-specific insertions. Alignment of pfHPPK-DHPS with HPPK and DHPS sequences from other microorganisms reveals two insertions relative to the corresponding enzyme in other organisms, i.e. HPPK-1 and HPPK-2. The former encompasses amino acid residues 66-162, while the latter covers residues 213-311. In order to investigate the roles of the two insertions, we constructed a number of mutants in which parts of these two insertions were deleted. Characterization of the mutationally altered proteins revealed that deletions of residues 74-80 in the HPPK-1 sequence of the pfHPPK-DHPS, but not that of the monofunctional pfHPPK, decreased the HPPK activity. A longer deletion (residues 74-86) in the HPPK-1 sequence of the bifunctional pfHPPK-DHPS completely inactivated both HPPK and DHPS activities. However, deletion in the HPPK-2 sequence from residues 247-306 did not disrupt the activities of HPPK and DHPS, but the kinetic properties of the recombinant proteins were slightly changed. The importance of HPPK-1 sequence on the catalytic activities of HPPK and DHPS in the bifunctional pfHPPK-DHPS could have implications for development of inhibitors targeting the non-catalytic region of this chemotherapeutically important enzyme.
authors.affiliation: Department of Biochemistry and Center for Bioinformatics and Applied Genomics, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
authors.forename: Woraphol
authors.initials: W
authors.lastname: Rattanachuen
authors.forename: Maria
authors.initials: M
authors.lastname: Jönsson
authors.forename: Göte
authors.initials: G
authors.lastname: Swedberg
authors.forename: Worachart
authors.initials: W
authors.lastname: Sirawaraporn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004094:Dihydropteroate Synthase / Q000235:genetics* / Q000378:metabolism*; D017854:Diphosphotransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D016415:Sequence Alignment; D017384:Sequence Deletion
chemical_list: D050505:Mutant Proteins; D004094:Dihydropteroate Synthase; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase