Plasmodium yoelii acetyl-coa carboxylase : detection and characterisation of the recombinant biotinoyl domain.

Abstract

Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design.