Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function

Newman, Adam G.; Vagstad, Anna L.; Belecki, Katherine; Scheerer, Jonathan R.; Townsend, Craig A.

doi:10.1039/c2cc36010a

Cited by 44 publications

(98 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All plasmids used in this study are summarized in Supplementary Table 3. The plasmids for expression of wild-type CTB1 SATKS-MAT (pECTB1-NKA6), PT (pECTB1-PT), and TE (pECTB1-TE) have been previously described 24 . Primers used for assembly of new plasmids in this study are detailed in Supplementary Table 4.…”

Section: Methodsmentioning

confidence: 99%

“…CTB1 ACP2 was cloned as an N-terminal Thrombin-cleavable His 6 −tagged construct to allow differential purification throughout the crosslinking process. The desired gene sequence was amplified by PCR from pECTB1-ACP (tandem ACP, previously described) 24 using CTB1-ACP2–5 and CTB1-ACP2-stop-3, and ligated into pET-28a at NdeI and NotI sites using T4 DNA ligase. Active site mutations for selective crosslinking were introduced into pECTB1-NKA6 by Gibson assembly 36 of PCR-amplified fragments using standard protocol, giving pECTB1-SKM-C119A-S1010A.…”

Section: Methodsmentioning

confidence: 99%

“…High-resolution mass data were collected on a Waters Acquity/Xevo-G2 in positive ion mode. Previously characterized CTB1 derailment products were identified by their UV-Vis spectrum and exact mass 24 .…”

Section: Methodsmentioning

confidence: 99%

“…For initiation of CTB1 biosynthesis, acetyl-CoA is loaded via the SAT 6 onto an ACP and transferred to the KS. The iterative action of the KS-MAT condensing region produces an ACP-tethered heptaketide (C14), which is subsequently cyclized by the PT and released as a pyrone by the TE 24 (Fig. 1).…”

mentioning

confidence: 99%

See 3 more Smart Citations

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…The "editing" behavior of TE domains is key to faithful and efficient synthesis [15,16]. So too are balanced internal kinetics of synthesis and transfer among active sites to maintain synthesis in the forward direction [15].…”

Section: Discussionmentioning

confidence: 99%

Deconstruction of Iterative Polyketide Synthases

Townsend¹

2014

New Chemistry and New Opportunities From the Expanding Protein Universe

Self Cite

View full text Add to dashboard Cite

The enormous progress of the last 25 years in natural products biochemistry has been dominated by investigation of giant microbial enzymes comprised of organized catalytic domains that function according to two distinct paradigms. Each is "programmed" to sequentially assemble simple precursor molecules over multiple steps without diffusible intermediates to synthesize highly elaborated products. Both synthetic strategies rely upon carrier proteins to hold and shuttle extending intermediates among catalytic domains in an ordered fashion. Collectively these megaproteins are impressive "supramolecular machines" exemplifying protein catalysis far more sophisticated than the classical view of "one gene, one enzyme, one reaction" [1], and are of central importance to the creation of small-molecule drugs for human and animal health. Assembly-line and iterative catalysis in natural product biosynthesisThe application of molecular biological techniques to natural product biosynthetic problems in the mid 1980's ushered in the modern era of research in the field. Seminal events include isolation of the penicillin gene cluster and characterization of the non-ribosomal peptide synthetase (NRPS) ACVS that assembles the classical Arnstein L,L,D-tripeptide precursor of the antibiotic [2], Fig. 1. Biochemical experiments on isolated wild-type NRPS enzymes had already established a linear mode of accreting ATP-dependent synthesis [3], but now isolation of the corresponding biosynthetic genes and their translation to domains of recognized function in loosely repeated sets, or "modules," underscored the exquisitely organized nature of the "assembly line" synthetic program executed by these large enzymes. The end of 1990 marked the watershed co-discovery of genes encoding three giant modular polyketide synthases (PKSs) that synthesize the 6-deoxyerythronolide core of the macrolide antibiotic erythromycin [4,5]. Together deoxyerythronolide B synthases (DEBS)1-3, totaling ca. 10 6 Da, opened the door to a second paradigm of assembly line megaenzymes whose synthetic program could be "read" from the N -to C-terminus.The parallel universes of NRPS and modular PKS enzymes in their canonical forms (there are many exceptions and variations on the theme) are extravagant extensions of the classical view of enzyme function enunciated 70 years ago [1]; that New Chemistry and New Opportunities from the Expanding Protein Universe Downloaded from www.worldscientific.com by NANYANG TECHNOLOGICAL UNIVERSITY on 08/20/15. For personal use only.

show abstract

Combinatorial Domain Swaps Provide Insights into the Rules of Fungal Polyketide Synthase Programming and the Rational Synthesis of Non‐Native Aromatic Products

et al. 2013

Self Cite

View full text Add to dashboard Cite

Nach den Regeln spielen: Der kombinatorische Domänenaustausch zwischen „abgebauten“ nichtreduzierenden Polyketid‐Synthasen (NR‐PKSs) deckt die Regeln auf, nach denen der Zusammenbau der Produkte erfolgt. Die von einzelnen katalytischen Domänen ausgeübte Kontrolle ist genügend groß, damit Heterokombinationen von Domänen unterschiedlicher NR‐PKSs Produkte in vorhersagbarer Weise synthetisieren.

show abstract

Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function

Abstract: The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases.

Cited by 44 publications

References 27 publications

The structural organization of substrate loading in iterative polyketide synthases

The structural organization of substrate loading in iterative polyketide synthases

Deconstruction of Iterative Polyketide Synthases

Combinatorial Domain Swaps Provide Insights into the Rules of Fungal Polyketide Synthase Programming and the Rational Synthesis of Non‐Native Aromatic Products

Contact Info

Product

Resources

About