Exploring Fungal Polyketide C-Methylation through Combinatorial Domain Swaps.

Polyketide C-methylation occurs during a programmed sequence of dozens of reactions carried out by multidomain polyketide synthases (PKSs). Fungal PKSs perform these reactions iteratively, where a domain may be exposed to and act upon multiple enzyme-tethered intermediates during biosynthesis. We surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs to gain insight into how different methylation patterns are installed. Our in vitro results show that control of methylation resides primarily with the CMeT, and CMeTs can intercept and methylate intermediates from noncognate nonreducing PKS domains. Furthermore, the methylation pattern is likely imposed by a competition between methylation or ketosynthase-catalyzed extension for each intermediate. Understanding site-specific polyketide C-methylation may facilitate targeted C-C bond formation in engineered biosynthetic pathways.

The structural organization of substrate loading in iterative polyketide synthases.

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

In trans hydrolysis of carrier protein-bound acyl intermediates by CitA during citrinin biosynthesis.

Polyketide synthases (PKSs) have several known editing mechanisms to ensure that non-productive intermediates are removed from the acyl carrier protein (ACP). We demonstrate that CitA, a putative hydrolase in the citrinin biosynthetic gene cluster, removes ACP-bound acyl intermediates. We propose that it serves an editing role in trans.

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin.

Fungal polyketide synthases (PKSs) are large, multidomain enzymes that biosynthesize a wide range of natural products. A hallmark of these megasynthases is the iterative use of catalytic domains to extend and modify a series of enzyme-bound intermediates. A subset of these iterative PKSs (iPKSs) contains a C-methyltransferase (CMeT) domain that adds one or more S-adenosylmethionine (SAM)-derived methyl groups to the carbon framework. Neither the basis by which only specific positions on the growing intermediate are methylated ("programming") nor the mechanism of methylation are well understood. Domain dissection and reconstitution of PksCT, the fungal non-reducing PKS (NR-PKS) responsible for the first isolable intermediate in citrinin biosynthesis, demonstrates the role of CMeT-catalyzed methylation in precursor elongation and pentaketide formation. The crystal structure of the S-adenosyl-homocysteine (SAH) coproduct-bound PksCT CMeT domain reveals a two-subdomain organization with a novel N-terminal subdomain characteristic of PKS CMeT domains and provides insights into co-factor and ligand recognition.

Systematic domain swaps of iterative, nonreducing polyketide synthases provide a mechanistic understanding and rationale for catalytic reprogramming.

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro "domain swapped" NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis.