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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 vitroreactions with Bref-PKS demonstrate the TH-controlledPKS chain length release. (A) EIC spectra of the in vitro reactionsshowed the variation in the product profiles of Bref-PKS with Bref-TH; Bref-PKS withbase hydrolysis; Bref-PKS with Bref-TH H276A; andBref-PKS with other in trans releasing enzymes CazEand Fma-AT. The reactions consist of 20 μM Bref-PKS, 2 mM mCoA,and 10 mM NADPH with either 20 μM concentration of the releasingenzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B)Proposed structures of 7 and 8.
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fig4: In vitroreactions with Bref-PKS demonstrate the TH-controlledPKS chain length release. (A) EIC spectra of the in vitro reactionsshowed the variation in the product profiles of Bref-PKS with Bref-TH; Bref-PKS withbase hydrolysis; Bref-PKS with Bref-TH H276A; andBref-PKS with other in trans releasing enzymes CazEand Fma-AT. The reactions consist of 20 μM Bref-PKS, 2 mM mCoA,and 10 mM NADPH with either 20 μM concentration of the releasingenzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B)Proposed structures of 7 and 8.

Mentions: More importantly, thetwo new products 7 (m/z 295 [M + H]+) and 8 (m/z 297 [M + H]+) were isolated from the aboveBref-PKS assay, with 7 now being the dominant productof all polyketide products (Figure 4A and Supplementary Figure S10). Both compounds displayedλmax of 215 nm, which hints at the absence of conjugateddienoic acid moiety. The +28 mu increase in masses of 7 and 8 compared to that of 3 and 5 suggests the incorporation of a completely reduced ketideunit (-CH2-CH2-) as a result of an additionalround of chain elongation and reduction by the Bref-PKS (Figure 4A and Supplementary Figure S10). Due to the single-turnover nature of the assay in the absenceof the Bref-TH, compounds 7 and 8 couldnot be sufficiently obtained for structural elucidation. Expressionof Bref-PKS alone in yeast did not lead to detectable amounts of 7 and 8 either. Therefore, to confirm compound 7 is indeed a nonaketide instead of octaketide, we performedthe in vitro assay using 2-13C-malonate and the MatB system,which generates the 2-13C-malonyl-CoA in situ.24 As expected, an increase of 9 mu in molecularweights was observed for both labeled 7 and 8, confirming the incorporation of nine ketide units derived frommalonate into the backbone (Supplementary FigureS8). Combining the UV and mass data (SupplementaryFigures S6 and S8), we propose the structure of 7 and 8 as shown in Figure 4B,derived from an additional round of chain elongation from 3 and 5, respectively, followed by full β-reduction.Selected ion monitoring of the mass of 7 and 8 in the Bref-PKS assay that contained Bref-TH yielded no trace ofthese two compounds. These results demonstrate that in the absenceof the Bref-TH, the Bref-PKS functions primarily as a nonaketide synthase.


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 vitroreactions with Bref-PKS demonstrate the TH-controlledPKS chain length release. (A) EIC spectra of the in vitro reactionsshowed the variation in the product profiles of Bref-PKS with Bref-TH; Bref-PKS withbase hydrolysis; Bref-PKS with Bref-TH H276A; andBref-PKS with other in trans releasing enzymes CazEand Fma-AT. The reactions consist of 20 μM Bref-PKS, 2 mM mCoA,and 10 mM NADPH with either 20 μM concentration of the releasingenzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B)Proposed structures of 7 and 8.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215887&req=5

fig4: In vitroreactions with Bref-PKS demonstrate the TH-controlledPKS chain length release. (A) EIC spectra of the in vitro reactionsshowed the variation in the product profiles of Bref-PKS with Bref-TH; Bref-PKS withbase hydrolysis; Bref-PKS with Bref-TH H276A; andBref-PKS with other in trans releasing enzymes CazEand Fma-AT. The reactions consist of 20 μM Bref-PKS, 2 mM mCoA,and 10 mM NADPH with either 20 μM concentration of the releasingenzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B)Proposed structures of 7 and 8.
Mentions: More importantly, thetwo new products 7 (m/z 295 [M + H]+) and 8 (m/z 297 [M + H]+) were isolated from the aboveBref-PKS assay, with 7 now being the dominant productof all polyketide products (Figure 4A and Supplementary Figure S10). Both compounds displayedλmax of 215 nm, which hints at the absence of conjugateddienoic acid moiety. The +28 mu increase in masses of 7 and 8 compared to that of 3 and 5 suggests the incorporation of a completely reduced ketideunit (-CH2-CH2-) as a result of an additionalround of chain elongation and reduction by the Bref-PKS (Figure 4A and Supplementary Figure S10). Due to the single-turnover nature of the assay in the absenceof the Bref-TH, compounds 7 and 8 couldnot be sufficiently obtained for structural elucidation. Expressionof Bref-PKS alone in yeast did not lead to detectable amounts of 7 and 8 either. Therefore, to confirm compound 7 is indeed a nonaketide instead of octaketide, we performedthe in vitro assay using 2-13C-malonate and the MatB system,which generates the 2-13C-malonyl-CoA in situ.24 As expected, an increase of 9 mu in molecularweights was observed for both labeled 7 and 8, confirming the incorporation of nine ketide units derived frommalonate into the backbone (Supplementary FigureS8). Combining the UV and mass data (SupplementaryFigures S6 and S8), we propose the structure of 7 and 8 as shown in Figure 4B,derived from an additional round of chain elongation from 3 and 5, respectively, followed by full β-reduction.Selected ion monitoring of the mass of 7 and 8 in the Bref-PKS assay that contained Bref-TH yielded no trace ofthese two compounds. These results demonstrate that in the absenceof the Bref-TH, the Bref-PKS functions primarily as a nonaketide synthase.

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