Expression, purification and crystallization of the Plasmodium falciparum enoyl reductase.
New hope has been gained in the control of the malaria parasite Plasmodium falciparum (pf) with the discovery that the parasite contains a prokaryotic type II fatty-acid synthase (FAS). Since enzymes of this type are absent in humans, they are potential targets for the development of new drugs. The enoyl reductase enzyme (ENR) belonging to this pathway is of particular interest because it has been shown to be inhibited by submicromolar concentrations of the antimicrobial agent triclosan. Here, the development of an efficient overexpression system for pfENR as a fusion protein with maltose-binding protein, its simple one-step purification and cleavage from its fusion protein and crystallization under new conditions with bound NAD(+) cofactor and triclosan are reported. The crystals belong to the space group P2(1), with approximate unit-cell parameters a = 88.2, b = 82.4, c = 94.8 A, beta = 90.77 degrees, and contain a tetramer in the asymmetric unit. Cryocooled crystals (100 K) diffracted to beyond 2.2 A resolution at the Daresbury Synchrotron Radiation Source.
Muench
Stephen P
SP
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, England.
Rafferty
John B
JB
McLeod
Rima
R
Rice
David W
DW
Prigge
Sean T
ST
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004791:Enzyme Inhibitors / Q000737:chemistry; D058619:Maltose-Binding Proteins; D009243:NAD / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010963:Plasmodium falciparum / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D014260:Triclosan / Q000737:chemistry; D014961:X-Ray Diffraction
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D014260:Triclosan; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)