Molecular and biochemical characterization of Toxoplasma gondii beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ).
Toxoplasma gondii, unlike its mammalian host, utilizes a type II fatty acid biosynthesis pathway in which the steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are good targets for the development of new therapeutic drugs directed against toxoplasmosis. In this report, we show by using reverse transcription-polymerase chain reaction analysis that beta-Hydroxyacyl-acyl carrier protein dehydratase (TgFABZ) is expressed both in tachyzoites and bradyzoites. Indirect immunofluorescence antibody test further shows the localization of TgFABZ protein in the apicoplast of both tachyzoites and bradyzoites. Enzyme dynamic analysis shows that the purified recombinant TgFABZ protein is soluble and active. The Km value of the enzyme for its substrate analog crotonoyl-CoA was estimated to be 82.57 +/- 10 microM.
Dautu
George
G
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Ueno
Akio
A
Munyaka
Biscah
B
Carmen
Gabriella
G
Makino
Souichi
S
Kobayashi
Yoshiyasu
Y
Igarashi
Makoto
M
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014122:Toxoplasma / Q000737:chemistry / Q000201:enzymology*
D000214:Acyl Coenzyme A; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C010701:crotonyl-coenzyme A; D006836:Hydro-Lyases; C036180:beta-hydroxyacyl-(acyl-carrier-protein)dehydrase