Characterization of Unexpected Self-Acylation Activity of Acyl Carrier Proteins in a Modular Type I Apicomplexan Polyketide Synthase

Abstract

Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylationor substrate acylation without an AT domainfor three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2–4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.

Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase

“…These self-acylating ACPs are capable of loading a large scope of CoA substrates onto their ppant arms without an AT domain, which is conventionally required for this activity. 49 The TgPKS2 putative release didomain is also unusual as it contains a KS and TR domain. Predominantly, a thioester reductase or thioesterase is preceded by an ACP domain.…”

“…The protein contains three defined modules and hypothetical loading and release didomains. Biochemical characterization of the AT and ACP domains revealed unexpected activities. , For instance, a unique self-acylation activity is observed with three of the four Tg PKS2 ACP domains ( Tg ACP2–4). These self-acylating ACPs are capable of loading a large scope of CoA substrates onto their ppant arms without an AT domain, which is conventionally required for this activity .…”

Examination of Secondary Metabolite Biosynthesis in Apicomplexa

“…Sequencing of Tg PKS2 revealed an 18.8 kb gene that encodes a single ∼680 kDa modular type I PKS (Figure 4A). [102] antiSMASH analysis of this protein indicates three distinct modules with multiple interesting features, including a hypothesized loading module devoid of a definitive AT domain. This domain arrangement (KS‐ACP) has been previously observed and characterized in a recently de‐orphanized Nocardia PKS where it was found that the loading KS domain had decarboxylative activity and modified malonyl‐ S ‐ACP to acetyl‐ S ‐ACP [103] .…”