Gunaratne, Ruwani; (1999) The N-myristoyltransferase gene from Plasmodium falciparum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Myristoyl Co A: N-myristoyltransferase (NMT) catalyses the transfer of the carbon 14 fatty acid from myristoyl-CoA to the N-terminal glycine of a variety of eukaryotic cellular and viral proteins. This occurs co-translationally i.e. as the protein is being synthesised or very soon after completion of protein synthesis. NMT has previously been cloned from the three pathogenic fungi: Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. Mutations which knock out the gene cause recessive lethality, suggesting that one or more N-myristoylated proteins is required for the viability of these pathogenic fungi. The purpose of this study was to identify and clone the Plasmodium falciparum NMT, then to express an enzymatically active, recombinant protein. A alignment of all known NMT amino acid sequences show areas of homology. Oligonucleotide primers were designed, based on these areas of homology and used to amplify part of Plasmodium falciparum NMT (PfNMT). Full length PfNMT was subsequently cloned using a series of genomic and vectorette libraries. The PfNMT gene covers 1617bp of genomic DNA and 1233bp of cDNA with 3 introns, all towards the 5'end of the gene. The human NMT possesses N-terminal extension, which is thought to direct the normally cytosolic enzyme to the free ribosomes. Extensive studies of the 5' end of PfNMT by PCR has confirmed the absence of this extension in the P. falciparum enzyme. PfNMT cDNA was expressed in the expression vector pTRC-HisC and the recombinant protein has an apparent molecular mass of 47kDa. Enzymatic activity of recombinant PfNMT was determined based on its ability to N-myristoylate a synthetic peptide in vitro. This assay confirmed that PfNMT is functionally active in vitro. Finally, the inhibition of both recombinant PfNMT and PfNMT from parasite cultures was investigated, for a comparison with the human NMT and to determine if inhibition of NMT from P. falciparum cultures was detrimental to the growth of the parasite.

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