A single amino acid residue controls acyltransferase activity in a polyketide synthase from Toxoplasma gondii

D’Ambrosio, Hannah K.; Ganley, Jack G.; Keeler, Aaron M.; Derbyshire, Emily R.

doi:10.1016/j.isci.2022.104443

Cited by 7 publications

(22 citation statements)

References 77 publications

(93 reference statements)

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“…gondii GT1 genome shows a putative type I PKS with multiple assembly gaps, which we have termed Tg PKS2. We previously resolved all AT sequences of Tg PKS2 using primer-based Sanger sequencing, but the full domain architecture remained unresolved . To address this, extensive primer-based Sanger sequencing efforts were conducted using T.…”

Section: Resultsmentioning

confidence: 99%

“…Tg PKS2 contains four predicted ACP domains (ACP1–4) (Figure S2). , The minimum boundaries of these domains were identified and each ACP was cloned into expression plasmids harboring a His 6 tag. All constructs were expressed in the E.…”

Section: Resultsmentioning

confidence: 99%

“…A free thiol on the ACP ppant arm is known to be critical for transacylation and self-acylation activity. ,, To probe the role of thiols in the observed Tg ACP2–4 self-acylation reaction, iodoacetamide was used to alkylate sulfhydryl groups of Tg ACP3 and then assess activity with mCoA using a previously described fluorescent coupled-enzyme assay. ,− Briefly, nucleophilic attack of CoA-thioesters releases free CoA-SH, which is coupled to the reduction of NAD+ to NADH. NADH formation is then continually monitored by fluorescence on a plate reader (Figure S5A–B).…”

Section: Resultsmentioning

confidence: 99%

“…A route to obtaining a mechanistic understanding of a PKS is through the isolation and characterization of its functional domains, where establishing substrate specificities offer clues to the final metabolite generated. To this end, we have previously demonstrated that 2 AT domains within Tg PKS2 are capable of malonyl-CoA hydrolysis in vitro , while wild-type AT3 was inactive (as a domain) or has a yet unknown substrate . In the current work, we sought to investigate the transfer of substrate from these AT domains to their cognate ACP domain.…”

Section: Discussionmentioning

confidence: 99%

“…Tg PKS2 is expressed in infectious T. gondii life stages, but its mechanism and the polyketide product(s) it produces are unknown …”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Tg PKS2 is expressed in infectious T. gondii life stages, but its mechanism and the polyketide product(s) it produces are unknown …”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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Examination of Secondary Metabolite Biosynthesis in Apicomplexa

2023

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Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome-sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of the polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.

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Crosstalk between primary and secondary metabolism: Interconnected fatty acid and polyketide biosynthesis in prokaryotes

West

Bailey

2023

Bioorganic & Medicinal Chemistry Letters

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A single amino acid residue controls acyltransferase activity in a polyketide synthase from Toxoplasma gondii

Cited by 7 publications

References 77 publications

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

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

Examination of Secondary Metabolite Biosynthesis in Apicomplexa

Crosstalk between primary and secondary metabolism: Interconnected fatty acid and polyketide biosynthesis in prokaryotes

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