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Fungal polyketide synthase product chain-length control by partnering thiohydrolase.

Zabala AO, Chooi YH, Choi MS, Lin HC, Tang Y - ACS Chem. Biol. (2014)

Bottom Line: Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds.Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction.Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.

ABSTRACT
Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.

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In vitro productsof Bref-PKS and Bref-TH are acyclic octaketideswith variable degrees of β-reduction. (A) HPLC and EIC traceof the in vitro reaction between Bref-PKS and Bref-TH. (B) Production of compounds 3–6 from the S. cerevisiae-NpgA strain co-expressingBref-PKS and Bref-TH. Notice the change in the production profilebetween days 1 and 3. The compounds were purified according to theirpeak production period. (C) Elucidated structures of compounds 3–6 from the corresponding NMR spectra.
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fig3: In vitro productsof Bref-PKS and Bref-TH are acyclic octaketideswith variable degrees of β-reduction. (A) HPLC and EIC traceof the in vitro reaction between Bref-PKS and Bref-TH. (B) Production of compounds 3–6 from the S. cerevisiae-NpgA strain co-expressingBref-PKS and Bref-TH. Notice the change in the production profilebetween days 1 and 3. The compounds were purified according to theirpeak production period. (C) Elucidated structures of compounds 3–6 from the corresponding NMR spectra.

Mentions: Most HRPKSs donot have a dedicated domain for product offloading, relying insteadon an in trans acyltransferase or TH to release thepolyketide from the ACP tether.2 Such partneringenzyme plays an important role in terminating the chain elongationand consequently in determining the length of the final product. Toinvestigate the influence of the in trans Bref-THon HRPKS function, the purified enzyme was added to the Bref-PKS in vitro reaction at an equimolar ratio (20 μM)with 2 mM malonyl-CoA and 10 mM NADPH. The reaction was left at RTfor 16 h and was extracted with ethyl acetate and analyzed by LC–MS.Four relatively hydrophobic compounds 3–6 emerged at the end of the gradient (5%–95% acetonitrilein H2O, 30 min), with 3 being the dominantproduct (Figure 3A). The corresponding massesfor compounds 3, 4, 5, and 6 are 264, 268, 264 and 266, respectively (Supplementary Figure S6). Compounds 3 and 5 displayed λmax of 260 nm, indicating thepresence of a slightly conjugated structure, while compounds 4 and 6 did not have significant absorption above220 nm. The masses of these compounds are consistent with that ofan octaketide that has undergone several reductive modifications (forreference, the molecular weight of palmitic acid is 256).


Fungal polyketide synthase product chain-length control by partnering thiohydrolase.

Zabala AO, Chooi YH, Choi MS, Lin HC, Tang Y - ACS Chem. Biol. (2014)

In vitro productsof Bref-PKS and Bref-TH are acyclic octaketideswith variable degrees of β-reduction. (A) HPLC and EIC traceof the in vitro reaction between Bref-PKS and Bref-TH. (B) Production of compounds 3–6 from the S. cerevisiae-NpgA strain co-expressingBref-PKS and Bref-TH. Notice the change in the production profilebetween days 1 and 3. The compounds were purified according to theirpeak production period. (C) Elucidated structures of compounds 3–6 from the corresponding NMR spectra.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215887&req=5

fig3: In vitro productsof Bref-PKS and Bref-TH are acyclic octaketideswith variable degrees of β-reduction. (A) HPLC and EIC traceof the in vitro reaction between Bref-PKS and Bref-TH. (B) Production of compounds 3–6 from the S. cerevisiae-NpgA strain co-expressingBref-PKS and Bref-TH. Notice the change in the production profilebetween days 1 and 3. The compounds were purified according to theirpeak production period. (C) Elucidated structures of compounds 3–6 from the corresponding NMR spectra.
Mentions: Most HRPKSs donot have a dedicated domain for product offloading, relying insteadon an in trans acyltransferase or TH to release thepolyketide from the ACP tether.2 Such partneringenzyme plays an important role in terminating the chain elongationand consequently in determining the length of the final product. Toinvestigate the influence of the in trans Bref-THon HRPKS function, the purified enzyme was added to the Bref-PKS in vitro reaction at an equimolar ratio (20 μM)with 2 mM malonyl-CoA and 10 mM NADPH. The reaction was left at RTfor 16 h and was extracted with ethyl acetate and analyzed by LC–MS.Four relatively hydrophobic compounds 3–6 emerged at the end of the gradient (5%–95% acetonitrilein H2O, 30 min), with 3 being the dominantproduct (Figure 3A). The corresponding massesfor compounds 3, 4, 5, and 6 are 264, 268, 264 and 266, respectively (Supplementary Figure S6). Compounds 3 and 5 displayed λmax of 260 nm, indicating thepresence of a slightly conjugated structure, while compounds 4 and 6 did not have significant absorption above220 nm. The masses of these compounds are consistent with that ofan octaketide that has undergone several reductive modifications (forreference, the molecular weight of palmitic acid is 256).

Bottom Line: Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds.Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction.Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.

ABSTRACT
Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.

Show MeSH
Related in: MedlinePlus