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 μM, Vmax = 2.236 μmol mg−1 min−1) and CoA (h = 0.704, K50 = 5.627 μM, Vmax = 0.557 μmol mg−1 min−1), and biphasic kinetics towards adenosine 5′-triphosphate (Km1 = 3.149 μM, Vmax1 = 373.3 nmol mg−1 min−1, Km2 = 121.0 μM, 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 μM). 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.