Analysis of the Ketosynthase-Chain Length Factor Heterodimer from the Fredericamycin Polyketide Synthase

Abstract

SUMMARY The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). The initiation module is thought to synthesize a C6 intermediate that is then transferred onto the elongation PKS module, which extends it into a C30 poly-β-ketoacyl product. Here we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologues, the fdm ketosynthase – chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 multiple mutants of the CLF were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over, but could initiate and at least partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologues.

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

“…This observation could reflect the limitation of relying on homology models built from a single, short-chain template structure, or it may suggest that the cavity size of the KS-CLFs encoding the largest polyketides does not expand enough to accommodate the entire nascent polyketide chain. It is possible that in the case of the longest polyketides (C28-C30), auxiliary enzyme(s) create an expanded solvent-excluded cage, which serves to protect reactive polyketide intermediates (5,6,12). Our clusterwide analysis reveals that CLF mutations are correlated with changes to the gene roster (discussed below).…”

“…2). We computationally tested the long-standing hypothesis that the volume of the KS-CLF amphipathic cavity, which houses the growing polyketide chain during biosynthesis, is a main determinant of polyketide chain length (5,(8)(9)(10)(11)(12)(13)(14)(15). Using the solved actinorhodin KS-CLF structure as a template for in silico mutagenesis, we calculated the predicted volume of the KS-CLF cavity for five PKS clades ( Fig.…”

“…Besides the ACP, other genes involved in backbone biosynthesis include acyltransferase (AT) and priming KS (KSIII). Most characterized type II systems are primed with acetyl units and "borrow" these enzymes from the FAS pathway (8)(9)(10)(11)(12)(13)(14)(15)(21)(22)(23). Systems using nonacetate starting units rely on secondary AT and KSIII enzymes (Fig.…”

“…The remarkable chemical diversity observed in this class of molecules is thought to originate from variations in chain length and tailoring reactions. Previous phylogenetic studies have revealed the role of the CLF in controlling the chain length of type II polyketides (8)(9)(10)(11)(12)(13)(14)(15), but the evolution of the KS-CLF within the context of the entire protein assembly is not well understood.…”

Analysis and Refactoring of the A-74528 Biosynthetic Pathway

“…First, as has been seen with other type II PKSs that have the capacity to synthesize very long chain products, auxiliary enzyme components can be presumed to modulate the chain length specificity of the san KS/CLF. 12,14 Second, although TW95a and TW95b are produced in small quantities by this strain, the product profile remains similar to JF77 (Figure S2), indicating that long chain polyketide formation is inefficient. Third, the lack of any identifiable hexadienyl-primed products indicates that priming of the minimal PKS by the initiation module is either inefficient, or does not occur at all in this construct.…”