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

Abstract

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.

Investigating A Multi-Domain Polyketide Synthase in Amphidinium carterae

“…This sequence was pulled from other multi-domain PKS, PKS/NRPS, and NRPS sequences because of a crucial correlation with toxicity [ 29 ]. The acyl-transferase (AT) domain was found in non-toxin-producing species but was missing in species that make toxins in a transcriptome survey on many dinoflagellate species ( Figure 3 ) [ 22 , 24 ]. This feature of AT domains not being included in type I PKSs is something that has been observed in other algae.…”

“…With an incre describing multi-domain PKS sequences obtained through transc cused on a triple-module PKS. This sequence was pulled from o PKS/NRPS, and NRPS sequences because of a crucial correlatio acyl-transferase (AT) domain was found in non-toxin-producing in species that make toxins in a transcriptome survey on many din ure 3) [22,24]. This feature of AT domains not being included in t that has been observed in other algae.…”

“…One multi-modular PKS termed the "Triple KS" for its three apparent ketosynthase modules and first described by Williams et al, is of particular interest in that it is ubiquitously present in dinoflagellate transcriptomes, indicating a core function [13,22]. Although the genes for classic lipid synthesis have been described elsewhere [23], other common processes that could be mediated by PKSs include PUFA synthesis and the "hairpin" structure that is found in many toxins across a wide range of Dinoflagellate species (Figure In addition to PKSs, non-ribosomal peptide synthetases (NRPS) and NRPS/PKS hybrids are also present in dinoflagellates [13].…”