Characterization of an Unusual α-Oxoamine Synthase Off-Loading Domain from a Cyanobacterial Type I Fatty Acid Synthase

Type I fatty acid synthases (FASs) are known from higher eukaryotes and fungi. We report the discovery of FasT, a rare type I FAS from the cyanobacterium Chlorogloea sp. CCALA695. FasT possesses an unusual off-loading domain, which was heterologously expressed in E. coli and found to act as an α-oxoamine synthase (AOS) in vitro. Similar to serine palmitoyltransferases from sphingolipid biosynthesis, the AOS off-loading domain catalyzes a decarboxylative Claisen condensation between l-serine and a fatty acyl thioester. While the AOS domain was strictly specific for l-serine, thioesters with saturated fatty acyl chains of six carbon atoms and longer were tolerated, with the highest activity observed for stearoyl−coenzyme A (C18). Our findings suggest a novel route to α-amino ketones via the direct condensation of iteratively produced long-chain fatty acids with l-serine by a FAS with a cis-acting AOS off-loading domain.ISSN:1439-4227ISSN:1439-763

Structure of a Promiscuous Thioesterase Domain Responsible for Branching Acylation in Polyketide Biosynthesis

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TEBs). Structural analysis of the oocydin (OocS) TEB domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TEB-mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TEB motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TEB and homologs as invaluable synthetic biology tools for polyketide drug development.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.ISSN:2568-225

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones

Enzymatic core components from trans‐acyltransferase polyketide synthases (trans‐AT PKSs) catalyze exceptionally diverse biosynthetic transformations to generate structurally complex bioactive compounds. Here we focus on a group of oxygenases identified in various trans‐AT PKS pathways, including those for pederin, oocydins, and toblerols. Using the oocydin pathway homologue (OocK) from Serratia plymuthica 4Rx13 and N‐acetylcysteamine (SNAC) thioesters as test surrogates for acyl carrier protein (ACP)‐tethered intermediates, we show that the enzyme inserts oxygen into β‐ketoacyl moieties to yield malonyl ester SNAC products. Based on these data and the identification of a non‐hydrolyzed oocydin congener with retained ester moiety, we propose a unified biosynthetic pathway of oocydins, haterumalides, and biselides. By providing access to internal ester, carboxylate pseudostarter, and terminal hydroxyl functions, oxygen insertion into polyketide backbones greatly expands the biosynthetic scope of PKSs.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Structural and Functional Studies of a Pyran Synthase Domain from a <i>trans</i>-Acyltransferase Assembly Line

<i>trans</i>-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized <i>cis</i>-acyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and six-membered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine

Automated structure prediction of trans-acyltransferase polyketide synthase products

ISSN:1552-4450ISSN:1552-446

Modular Oxime Formation by a trans-AT Polyketide Synthase

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Evolution-Guided Engineering of Trans-Acyltransferase Polyketide Synthases

&lt;p&gt;Data underlying the manuscript 'Evolution-Guided Engineering of &lt;em&gt;Trans&lt;/em&gt;-Acyltransferase Polyketide Synthases' by Mabesoone, Leopold-Messer and Minas et al.&lt;/p&gt; &lt;p&gt;The repository contains:&lt;/p&gt; &lt;p&gt;Sequencing data - Genbanks files of construct designs, ab1 and fasta files for Sanger sequencing. For whole plasmid sequencing, fasta, annotated GenBank files, overviews of sequencing statistics and fastq files for selected plasmids, for which fastq files were provided by the sequencing service, are provided.&lt;/p&gt; &lt;p&gt;NMR data - raw data and MestReNova files. Also includes HPLC-MS traces of isolated compounds.&lt;/p&gt; &lt;p&gt;HPLC-MS data - Raw data collected on Thermo-Fisher instruments. This data can be analyzed with the Xcalibur software suite. The mzXML data can be analyzed with the Python scripts provided in the scripts folder to generate the images shown in the SI. The data collected for Bacillus and Serratia is MS1 data. The data collected for Gynuella also contains MS-MS data.&lt;/p&gt; &lt;p&gt;SCA data - Python scripts, GenBank files and produced data underlying the SCA.&lt;/p&gt; &lt;p&gt;Bioactivity data - Data underlying the toxicity assays in Figure S122.&lt;/p&gt; &lt;p&gt;&nbsp;&lt;/p&gt

Modular Halogenation, α-Hydroxylation, and Acylation by a Remarkably Versatile Polyketide Synthase

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083