Employing 25-Residue Docking Motifs from Modular Polyketide Synthases as Orthogonal Protein Connectors.

The proteins of -acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of ∼25 residues at the C- and N-termini of these polypeptides (DDs and DDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.

Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line.

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cis-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.

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.

Portability and Structure of the Four-Helix Bundle Docking Domains of trans-Acyltransferase Modular Polyketide Synthases.

The polypeptides of multimodular polyketide synthases self-assemble into biosynthetic factories. While the docking domains that mediate the assembly of cis-acyltransferase polyketide synthase polypeptides are well-studied, those of the more recently discovered trans-acyltransferase polyketide synthases have just started to be described. Located at the C- and N-termini of many polypeptides, these 25-residue, two-helix, pseudosymmetric motifs noncovalently connect domains both between and within modules.

α-Methylation follows condensation in the gephyronic acid modular polyketide synthase.

C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked β-ketoacylthioester substrates but not malonyl thioester equivalents.

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.

The structure of SpnF, a standalone enzyme that catalyzes [4 + 2] cycloaddition.

In the biosynthetic pathway of the spinosyn insecticides, the tailoring enzyme SpnF performs a [4 + 2] cycloaddition on a 22-membered macrolactone to forge an embedded cyclohexene ring. To learn more about this reaction, which could potentially proceed through a Diels-Alder mechanism, we determined the 1.50-Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine.

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.

Investigating the reactivities of a polyketide synthase module through fluorescent click chemistry.

A method for monitoring in vitro polyketide synthesis has been developed whereby nonchromophoric polyketide products are made brightly fluorescent in a simple, rapid, inexpensive, and bioorthogonal manner through CuAAC with a sulforhodamine B azide derivative.