Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase.
The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.
Fritzler
Jason M
JM
Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA.
Zhu
Guan
G
D000213:Acyl Carrier Protein / Q000502:physiology*; D020816:Amino Acid Motifs; D000818:Animals; D001426:Bacterial Proteins / Q000502:physiology; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D008025:Ligases / Q000502:physiology*; D048630:Polyketide Synthases / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000502:physiology
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D048630:Polyketide Synthases; D017855:Transferases (Other Substituted Phosphate Groups); D008025:Ligases