Over-production of lactate dehydrogenase from Plasmodium falciparum opens a route to new antimalarials
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Over-production of lactate dehydrogenase (PfLDH) from Plasmodium falciparum from E. coli TG2 cells transformed with a pKK223-3 plasmid containing the wild type gene isolated by Bzik DJ, Fox BA, and Gonyer K (1993) Mol. Biochem. Parasit.59, 155–166, gave mostly an inactive protein after isolation. Sequencing the N-terminus of the over-produced protein showed that the major product commenced at an internal methionine. Truncation of the protein occurred due to the inappropriate priming from a Shine–Dalgarno (SD) sequence upstream of Met 35. Silent mutations of this SD sequence to remove the purine-rich region allowed over-production of the full length PfLDH up to 15 mg protein l−1 broth. The purified protein exhibited biochemical properties of an authentic LDH enzyme. However, high activity with 3-acetylpyridine adenine dinucleotide as well as with the natural cofactor, NAD, was also observed. The high-resolution X-ray structure obtained from the recombinant enzyme has provided the opportunity for the development of inhibitors specific to PfLDH.
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- Barstow DA, Clarke AR, Chia WN, Wigley DB, Sharman AF, Holbrook JJ, Atkinson T, Minton P (1986) Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene. Gene 46: 47–55.Google Scholar
- Bzik DJ, Fox BA, Gonyer K (1993) Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli. Mol. Biochem. Parasit. 59: 155–166.Google Scholar
- Dunn CR, Banfield MJ, Barker JJ, Higham CW, Moreton, KM, Turgut-Balik D, Brady RL, Holbrook JJ (1996) The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for antimalarial design. Nature Struct. Biol. 3: 912–915.Google Scholar
- Eszes CM, Sessions RB, Clarke AR, Moreton KM, Holbrook JJ (1996) Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change. FEBS Lett. 399: 193–197.Google Scholar
- Kim JH, Pack MY (1993) Over-production of extracellular endoglucanase by genetically engineered Bacillus subtilis. Biotechnol. Lett. 15: 133–138.Google Scholar
- Makler MT, Hinrichs DJ (1993) Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am. J. Trop. Med. Hyg. 48: 205–210.Google Scholar
- Royer RE, Deck LM, Campos NM, Hunsaker LA, Vander Jagt DL (1986) Biologically active derivatives of gossypol. Synthesis and antimalarial activities of peri-acylated gossylic nitriles. J. Med. Chem. 29: 1799–1801.Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
- Sessions RB, Dewar V, Clarke AR, Holbrook JJ (1997) A model of the Plasmodium falciparum lactate dehydrogenase and its implications for the design of new antimalarials. Protein Eng. 10: 301–306.Google Scholar
- Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71: 1342–1346.Google Scholar
- White JL, Hackert ML, Buehner M, Adams MJ, Ford GC, Lentz PJ, Smiley IE, Steiner SJ, Rossman MG (1976) A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes. J. Mol. Biol. 102: 759–779.Google Scholar