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Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

ABSTRACT: Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

SUBMITTER: Wang F

PROVIDER: S-EPMC3514014 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

Wang Flora F Krai Priscilla P Deu Edgar E Bibb Brittney B Lauritzen Conni C Pedersen John J Bogyo Matthew M Klemba Michael M

Molecular and biochemical parasitology 20100915 1

Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride- ...[more]

PMID: 20833209

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Project description:Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase ? is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme.The coding sequences of DNA polymerase ? catalytic subunit (PfPol?-cat), DNA polymerase ? small subunit (PfPol?S) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPol?-cat was biochemically characterized. The roles of PfPol?S and PfPCNA in PfPol?-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPol?-cat activity and on in vitro parasite growth using SYBR Green I assay.The purified recombinant protein PfPol?-cat, PfPol?S and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPol?-cat and PfPol?S had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPol?-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt (>200 mM). PfPol?S stimulated PfPol?-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPol?-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 µM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 µM).Recombinant PfPol?-cat, PfPol?S and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPol?-cat. The high sensitivity of PfPol? to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.

Project description:BACKGROUND: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. METHODS: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ?folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. RESULTS: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. CONCLUSIONS: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor.

Dataset Information

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

Publications

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

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