The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line.

Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae.

The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast.

Divergence of multimodular polyketide synthases revealed by a didomain structure.

The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 Å-resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ∼600-Å(2) interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues.

Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases.

In an effort to uncover the structural motifs and biosynthetic logic of the relatively uncharacterized trans-acyltransferase polyketide synthases, we have begun the dissection of the enigmatic dehydrating bimodules common in these enzymatic assembly lines. We report the 1.98 Å resolution structure of a ketoreductase (KR) from the first half of a type A dehydrating bimodule and the 2.22 Å resolution structure of a dehydratase (DH) from the second half of a type B dehydrating bimodule. The KR, from the third module of the bacillaene synthase, and the DH, from the tenth module of the difficidin synthase, possess features not observed in structurally characterized homologs. The DH architecture provides clues for how it catalyzes a unique double dehydration. Correlations between the chemistries proposed for dehydrating bimodules and bioinformatic analysis indicate that type A dehydrating bimodules generally produce an α/ß-cis alkene moiety, while type B dehydrating bimodules generally produce an α/ß-trans, γ/δ-cis diene moiety.

A double-hotdog with a new trick: structure and mechanism of the trans-acyltransferase polyketide synthase enoyl-isomerase.

Many polyketide natural products exhibit invaluable medicinal properties, yet much remains to be understood regarding the machinery responsible for their biosynthesis. The recently discovered trans-acyltransferase polyketide synthases employ processing enzymes that catalyze modifications unique from those of the classical cis-acyltransferase polyketide synthases. The enoyl-isomerase domains of these megasynthases shift double bonds and are well-represented by an enzyme that helps forge the triene system within the antibiotic produced by the prototypical bacillaene synthase. This first crystal structure of an enoyl-isomerase, at 1.73 Å resolution, not only revealed relationships between this class of enzymes and dehydratases but also guided an investigation into the mechanism of double bond migration. The catalytic histidine, positioned differently from that of dehydratases, was demonstrated to independently shuttle a proton between the γ- and α-positions of the intermediate. This unprecedented mechanism highlights the catalytic diversity of divergent enzymes within trans-acyltransferase polyketide synthases.

A close look at a ketosynthase from a trans-acyltransferase modular polyketide synthase.

The recently discovered trans-acyltransferase modular polyketide synthases catalyze the biosynthesis of a wide range of bioactive natural products in bacteria. Here we report the structure of the second ketosynthase from the bacillaene trans-acyltransferase polyketide synthase. This 1.95 Å resolution structure provides the highest resolution view available of a modular polyketide synthase ketosynthase and reveals a flanking subdomain that is homologous to an ordered linker in cis-acyltransferase modular polyketide synthases. The structure of the cysteine-to-serine mutant of the ketosynthase acylated by its natural substrate provides high-resolution details of how a native polyketide intermediate is bound and helps explain the basis of ketosynthase substrate specificity. The substrate range of the ketosynthase was further investigated by mass spectrometry.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.