Kira J. Weissman

Evaluating Ketoreductase Exchanges as a Means of Rationally Altering Polyketide Stereochemistry

Modular polyketide synthases (PKSs) are multidomain multienzymes responsible for the biosynthesis in bacteria of a wide range of polyketide secondary metabolites of clinical value. The stereochemistry of these molecules is an attractive target for genetic engineering in attempts to produce analogues exhibiting novel therapeutic properties. The exchange of ketoreductase (KR) domains in model PKSs has been shown in several cases to predictably alter the configuration of the β‐hydroxy functionalities but not of the α‐methyl groups. By systematic screening of a broad panel of KR domains, we have identified two donor KRs that afford modification of α‐methyl group stereochemistry. To the best of our knowledge, this provides the first direct in vivo evidence of KR‐catalyzed epimerization. However, none of the introduced KRs afforded simultaneous alteration of methyl and hydroxy configurations in high yield. Therefore, swapping of whole modules might be necessary to achieve such changes in stereochemistry.

Insights into the function of trans-acyl transferase polyketide synthases from the SAXS structure of a complete module

The modular polyketide synthases (PKS) are gigantic multienzymes which synthesize diverse secondary metabolites of therapeutic value. Although structural information is increasingly available for the ‘cis-AT’ class of modular PKS, almost nothing is known about the evolutionarily divergent ‘trans-AT’ PKS, which characteristically incorporate an iteratively-acting acyl transferase. We report here the SAXS solution structure of a complete apo module from the virginiamycin trans-AT PKS, which is fundamentally different to structural models proposed for the cis-AT PKS based on the crystal structure of animal fatty acid synthase. The module, which serves as a platform for the β-modification of the polyketide intermediate, consists of a ketosynthase (KS) and two acyl carrier protein (ACP) domains. In our solved structure, the homodimeric KS, which is flanked by well-folded linker regions, occupies the center of the module. While the first ACP is located close to the KS, the second is situated at the end of a flexible linker, and mobile. Taken together, these data provide a physical explanation for the functional non-equivalence previously observed for certain tandem ACPs of trans-AT PKS. Furthermore, the overall open shape of the module renders the second ACP highly accessible, which may be critical for its interaction with multiple in trans catalytic partners. Finally, our analysis redefines the function of a putative dimerization motif of tandem ACPs as a docking domain, suggesting that the module likely adopts a more closed form in order to affect transfer of the chain extension intermediate to the subsequent module.

Polyketide stereocontrol: a study in chemical biology

The biosynthesis of reduced polyketides in bacteria by modular polyketide synthases (PKSs) proceeds with exquisite stereocontrol. As the stereochemistry is intimately linked to the strong bioactivity of these molecules, the origins of stereochemical control are of significant interest in attempts to create derivatives of these compounds by genetic engineering. In this review, we discuss the current state of knowledge regarding this key aspect of the biosynthetic pathways. Given that much of this information has been obtained using chemical biology tools, work in this area serves as a showcase for the power of this approach to provide answers to fundamental biological questions.

Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis

Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.The monoamine oxidase family member LkcE is an enzyme from the lankacidin polyketide biosynthetic pathway, where it catalyzes an amide oxidation followed by an intramolecular Mannich reaction, yielding the polyketide macrocycle. Here the authors characterize LkcE and present several of its crystal structures, which explains the unusual dual activity of LkcE.

Probing Interactions in Fungal PKS

Biosynthesis of polyketides can depend on interactions between the acyl carrier proteins (ACPs) which hold the growing chains and their enzymatic partners. In this issue of Chemistry & Biology, Bruegger and colleagues demonstrate that mechanism-based probes tethered to the ACPs of fungal nonreducing polyketide synthases can provide insights into these contacts.