Identification, characterization, and inhibition of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ).
The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a Km of 199 microM and kcat/Km of 80.4 m-1 s-1 for the substrate analog beta-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (Km = 86 microM, kcat/Km = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli beta-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria.
Sharma
Shailendra Kumar
SK
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Kapoor
Mili
M
Ramya
T N C
TN
Kumar
Sanjay
S
Kumar
Gyanendra
G
Modak
Rahul
R
Sharma
Shilpi
S
Surolia
Namita
N
Surolia
Avadhesha
A
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002474:Cell-Free System; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004926:Escherichia coli / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013816:Thermodynamics
D005227:Fatty Acids; D006836:Hydro-Lyases; C036180:beta-hydroxyacyl-(acyl-carrier-protein)dehydrase