Phosphopantetheinylation in the green microalgae Chlamydomonas reinhardtii

Sonnenschein, Eva C.; Pu, Yuan; Beld, Joris; Burkart, Michael D.

doi:10.1007/s10811-016-0875-7

Cited by 4 publications

(6 citation statements)

References 56 publications

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“…The E. coli colonies were allowed to induce expression and produce indigoidine for 48 h, and, not shown here due to a loss of quantitation, would eventually become dark in color for most insert/activator combinations. This immediately leads to the conclusion that all forms of the reporter are capable of producing indigoidine and that there is some possible phosphopantethienation by each dinoflagellate PPTase for any given thiolation domain, another example of promiscuous PPTase binding in protists [49]. This also verifies the prediction that these enzymes transfer the phosphopantetheinate group and not other moieties, as has been shown in rare cases [50].…”

Section: Discussionsupporting

confidence: 67%

“…The rationale is that, if a given phosphopantetheinyl transferase can activate the thiolation domain of the BpsA reporter, then indigoidine will be produced. This pairing of activator and thiolation domain is a common method for determining specificity [46,47] and has been performed in some protists with a surprising promiscuity not found in bacteria and fungi [48,49]. This study advances previous work by replacing the thiolation domain of the BpsA reporter with several different dinoflagellate sequences to allow for the pairing of each activator with a multitude of potential phosphopantetheination sites.…”

Section: Introductionmentioning

confidence: 84%

“…When considering the phosphopantetheinyl transferases (PPTases) that activate these thiolation domains, which have fewer than three types and a low copy number [43], the number of combinations becomes tractable for biochemical analysis. Thus, this study aims to begin a bottom-up approach, where the specificity of a PPTase for a particular pathway, vis-à-vis that pathway's thiolation domains, can be exploited to isolate toxin synthesis pathways once that specificity has been identified, although there appears to be some unusual promiscuity in protists [49].…”

Section: Discussionmentioning

confidence: 99%

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Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

2022

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Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward.

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Section: Discussionsupporting

confidence: 67%

Section: Introductionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

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Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

2022

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“…43,44 In our work on the discovery of inhibitors for the B. subtilis PPTase, Sfp, we found the general PPTase inhibitor 6-NOBP 5 shows modest inhibition against a variety of PPTases. 43,45 Based on these findings Owen et al 14 The fourth domain of BpsA is the TE, which presumably releases the product (or an intermediate) from the PCP. While the exact mechanism is unknown, release of 5-amino-pyridinedione, followed by spontaneous dimerization of two products, forming either colorless leuco-indigoidine or blue indigoidine, is a proposed mechanism.…”

mentioning

confidence: 99%

“…PPTase inhibitor 5 shows toxicity, but no clear absence of blue pigmentation, perhaps because 5 is a an unspecific PPTase inhibitor and E. coli harbors an essential PPTase that is also inhibited by 6-NOBP. 45 The thioesterase inhibitors 4 and ABESF show severe toxicity to E. coli at high concentrations. At lower concentrations, both general inhibitors still exhibited toxicity, but the presence of white colonies could be detected (Fig.…”

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confidence: 99%

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach

Vickery

McCulloch

Sonnenschein

et al. 2020

Bioorganic & Medicinal Chemistry Letters

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A Comparison of Dinoflagellate Thiolation Domain Binding Proteins Using In Vitro and Molecular Methods

2022

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Dinoflagellates play important roles in ecosystems as primary producers and consumers making natural products that can benefit or harm environmental and human health but are also potential therapeutics with unique chemistries. Annotations of dinoflagellate genes have been hampered by large genomes with many gene copies that reduce the reliability of transcriptomics, quantitative PCR, and targeted knockouts. This study aimed to functionally characterize dinoflagellate proteins by testing their interactions through in vitro assays. Specifically, nine Amphidinium carterae thiolation domains that scaffold natural product synthesis were substituted into an indigoidine synthesizing gene from the bacterium Streptomyces lavendulae and exposed to three A. carterae phosphopantetheinyl transferases that activate synthesis. Unsurprisingly, several of the dinoflagellate versions inhibited the ability to synthesize indigoidine despite being successfully phosphopantetheinated. However, all the transferases were able to phosphopantetheinate all the thiolation domains nearly equally, defying the canon that transferases participate in segregated processes via binding specificity. Moreover, two of the transferases were expressed during growth in alternating patterns while the final transferase was only observed as a breakdown product common to all three. The broad substrate recognition and compensatory expression shown here help explain why phosphopantetheinyl transferases are lost throughout dinoflagellate evolution without a loss in a biochemical process.

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Phosphopantetheinylation in the green microalgae Chlamydomonas reinhardtii

Cited by 4 publications

References 56 publications

Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

Dissecting modular synthases through inhibition: A complementary chemical and genetic approach

A Comparison of Dinoflagellate Thiolation Domain Binding Proteins Using In Vitro and Molecular Methods

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