Recognition of Intermediate Functionality by Acyl Carrier Protein over a Complete Cycle of Fatty Acid Biosynthesis

Płoskoń, Eliza; Arthur, Christopher J.; Kanari, Amelia L.P.; Wattana-Amorn, Pakorn; Williams, Christopher; Crosby, John; Simpson, Thomas J.; Willis, Christine L.; Crump, Matthew P.

doi:10.1016/j.chembiol.2010.05.024

Cited by 50 publications

(59 citation statements)

References 38 publications

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“…9,27 Acyl-ACP species have been shown to negotiate client domain interactions in modular PKSs and FASs. 42,43 Indeed, in some of the combinatorial reactions containing the CTB1 and ACAS PT domains, the noncognate PT influenced the final chain length, directing formation of a longer or shorter intermediate, respectively.…”

Section: Discussionmentioning

confidence: 99%

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

et al. 2014

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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.

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Section: Discussionmentioning

confidence: 99%

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

et al. 2014

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“…Additional TE contacts occur with the C terminus of helix 1 in the carrier domains (22). In light of the results from the Ent system, the structures of FabC (44) and AcpP (45) are informative about the basis of the ACP selectivity of RedJ. The surface of the helix 3 C terminus is positively charged in FabC and negatively charged in RedQ and AcpP (supplemental Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Whicher

Florova

Sydor

et al. 2011

Journal of Biological Chemistry

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Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a ␤-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.Modular polyketide synthases (PKSs) 5 and non-ribosomal peptide synthetases (NRPSs) are large multienzymes that catalyze the production of a wide range of biologically active compounds currently used as therapies for human diseases (1). Most PKSs and NRPSs are organized as assembly lines in which specific enzymatic domains catalyze elongation or modification of specific pathway intermediates. One key difference between these two types of assembly lines is that PKSs use acyl thioesters as substrates, whereas NRPSs use amino acids. Both PKS and NRPS pathway intermediates are tethered, via a thioester bond, to the phosphopantetheine (Ppant) arms of acyl/ peptidyl carrier protein (ACP/PCP) domains throughout chain assembly. Thioester cleavage, most often catalyzed by a terminating or type I thioesterase (TE I), releases the fully assembled polyketide/peptide from the carrier domain.Prodiginine biosynthesis is directed by the red cluster in Streptomyces coelicolor and involves hybrid NRPS/PKS multienzymes employing an ␣-oxoamine synthase (OAS) in a highly unusual chain release mechanism to assemble undecylprodiginine (1) and its carbocyclic derivative streptorubin B (2) (Fig. 1A) (2-5). The therapeutic potential of prodiginines was demonstrated in prior studies in which analogues of undecylprod...

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“…Biosynthetic intermediates with varied chemical structures participate in intermolecular interactions with ACP that modulate substrate dynamics and ACP structure 9,15 . The nature of these ACP-substrate interactions depends on the chemical structure of the biosynthetic intermediate and can vary with respect to chain length and oxidation state 23 . Furthermore, observations of this phenomenon appear to vary depending on the analytical method used.…”

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confidence: 99%

Reversible labeling of native and fusion-protein motifs

et al. 2012

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The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of E. coli acyl carrier protein (ACP) for visualization and functional studies. We utilize this iterative enzymatic methodology in vitro for reversible labeling variants and apply these tools to Nuclear Magnetic Resonance (NMR) structural studies of protein-substrate interactions.

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Recognition of Intermediate Functionality by Acyl Carrier Protein over a Complete Cycle of Fatty Acid Biosynthesis

Cited by 50 publications

References 38 publications

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Reversible labeling of native and fusion-protein motifs

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