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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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Fma-PKS produces longer polyenes in theabsence of cognate Fma-AT.(A) HPLC profiles of Fma-PKS with Fma-AT; base hydrolysis; or Bref-TH.Fma-PKS produces a hexaketide polyene in the presence of the partnerFma-AT. In its absence or in the presence of the non-cognate Bref-TH,the PKS catalyzes 1 or 2 more extension steps to yield the heptaketide 10 and octaketide 11. The reactions consist of20 μM Fma-PKS, 2 mM mCoA, and 2 mM NADPH with either 20 μMof the releasing enzyme or base hydrolysis (1 M NaOH at 65 °Cfor 10 min). (B) Proposed structures of the polyene compounds producedin the in vitro assay.
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fig5: Fma-PKS produces longer polyenes in theabsence of cognate Fma-AT.(A) HPLC profiles of Fma-PKS with Fma-AT; base hydrolysis; or Bref-TH.Fma-PKS produces a hexaketide polyene in the presence of the partnerFma-AT. In its absence or in the presence of the non-cognate Bref-TH,the PKS catalyzes 1 or 2 more extension steps to yield the heptaketide 10 and octaketide 11. The reactions consist of20 μM Fma-PKS, 2 mM mCoA, and 2 mM NADPH with either 20 μMof the releasing enzyme or base hydrolysis (1 M NaOH at 65 °Cfor 10 min). (B) Proposed structures of the polyene compounds producedin the in vitro assay.

Mentions: To examine whether chain-length regulationby the releasing enzyme is also observed in other HRPKSs, we assayedthe Fma-PKS involved in the biosynthesis of the polyene portion ofthe meroterpenoid fumagillin. Fma-PKS was previously shown to producethe highly conjugated hexaketide pentaenoic acid 9 ([M+ H+] 191, λmax 358 nm) in the presenceof its releasing acyltransferase partner, Fma-AT.28 When the Fma-PKS assay was performed in the absence ofFma-AT and subjected to base-hydrolysis followed by extraction, twonew products were observed, 10 ([M + H] + 217)and 11 ([M + H] + 243), with λmax of 378 and 398 nm, respectively. The periodic increases in bothmass (+26 mu) and λmax compared to 9 therefore strongly indicate 10 and 11 areheptaketide and octaketide polyenes, respectively, as shown in Figure 5. This is also verified by the in vitro labelingstudies with 2-13C-malonate that showed the correspondingmass shift of +7 and +8 for 10 and 11, respectively(Supplementary Figure S12 and S13). WhenBref-TH is used in the reaction with Fma-PKS, 10 and 11 were also dominantly produced relative to 9 (Figure 5A).


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

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

Fma-PKS produces longer polyenes in theabsence of cognate Fma-AT.(A) HPLC profiles of Fma-PKS with Fma-AT; base hydrolysis; or Bref-TH.Fma-PKS produces a hexaketide polyene in the presence of the partnerFma-AT. In its absence or in the presence of the non-cognate Bref-TH,the PKS catalyzes 1 or 2 more extension steps to yield the heptaketide 10 and octaketide 11. The reactions consist of20 μM Fma-PKS, 2 mM mCoA, and 2 mM NADPH with either 20 μMof the releasing enzyme or base hydrolysis (1 M NaOH at 65 °Cfor 10 min). (B) Proposed structures of the polyene compounds producedin the in vitro assay.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Fma-PKS produces longer polyenes in theabsence of cognate Fma-AT.(A) HPLC profiles of Fma-PKS with Fma-AT; base hydrolysis; or Bref-TH.Fma-PKS produces a hexaketide polyene in the presence of the partnerFma-AT. In its absence or in the presence of the non-cognate Bref-TH,the PKS catalyzes 1 or 2 more extension steps to yield the heptaketide 10 and octaketide 11. The reactions consist of20 μM Fma-PKS, 2 mM mCoA, and 2 mM NADPH with either 20 μMof the releasing enzyme or base hydrolysis (1 M NaOH at 65 °Cfor 10 min). (B) Proposed structures of the polyene compounds producedin the in vitro assay.
Mentions: To examine whether chain-length regulationby the releasing enzyme is also observed in other HRPKSs, we assayedthe Fma-PKS involved in the biosynthesis of the polyene portion ofthe meroterpenoid fumagillin. Fma-PKS was previously shown to producethe highly conjugated hexaketide pentaenoic acid 9 ([M+ H+] 191, λmax 358 nm) in the presenceof its releasing acyltransferase partner, Fma-AT.28 When the Fma-PKS assay was performed in the absence ofFma-AT and subjected to base-hydrolysis followed by extraction, twonew products were observed, 10 ([M + H] + 217)and 11 ([M + H] + 243), with λmax of 378 and 398 nm, respectively. The periodic increases in bothmass (+26 mu) and λmax compared to 9 therefore strongly indicate 10 and 11 areheptaketide and octaketide polyenes, respectively, as shown in Figure 5. This is also verified by the in vitro labelingstudies with 2-13C-malonate that showed the correspondingmass shift of +7 and +8 for 10 and 11, respectively(Supplementary Figure S12 and S13). WhenBref-TH is used in the reaction with Fma-PKS, 10 and 11 were also dominantly produced relative to 9 (Figure 5A).

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