Limits...
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.

Show MeSH

Related in: MedlinePlus

Putative biosynthethicpathway for 1. The HRPKS isproposed to synthesize the precisely reduced octaketide precursor,which could then be directly offloaded by the thiohydrolase enzymefollowed by a P450-mediated formation of the cyclopentane ring andmacrocyclization to afford the 7-deoxy BFA 2 (top scheme).Alternatively, the first ring annulation can also occur on the ACP-tetheredintermediate before the thiohydrolase release and lactonization (bottomscheme). The C7-hydroxylation is believed to be the final step inthe process to obtain the final structure of 1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215887&req=5

fig1: Putative biosynthethicpathway for 1. The HRPKS isproposed to synthesize the precisely reduced octaketide precursor,which could then be directly offloaded by the thiohydrolase enzymefollowed by a P450-mediated formation of the cyclopentane ring andmacrocyclization to afford the 7-deoxy BFA 2 (top scheme).Alternatively, the first ring annulation can also occur on the ACP-tetheredintermediate before the thiohydrolase release and lactonization (bottomscheme). The C7-hydroxylation is believed to be the final step inthe process to obtain the final structure of 1.

Mentions: In this study, we chose the HRPKS responsiblefor brefeldin A (BFA)as a model system (Figure 1). BFA (1) is a protein-transport inhibitor isolated from several speciesof filamentous fungi.10−14 It is used to study protein transport among eukaryotes but has alsobeen found to have antiviral, antifungal, and antitumor properties.15 The polyketide origin of this 16-membered macrolactonewas previously established through feeding studies with labeled acetate.16−18 The proposed biosynthesis of BFA involves formation of an acyclicpolyketide chain that is differentially tailored throughout the backbone(Figure 1). The presence of the terminal hydroxylgroup, along with the strategically positioned double bonds, is proposedto enable cyclization of the acyclic precursor into the fused, bicyclicstructure seen in BFA. We hypothesized that a single HRPKS shouldbe sufficient to generate the entire carbon backbone without the needfor an additional PKS. If the acyclic product indeed contains conjugateddouble bonds as proposed, the biosynthetic product should be readilyidentifiable and isolated. Collectively, the BFA HRPKS appears tofit the mold as a good model HR-PKS for biochemical analysis. Here,we first identified the HRPKS most likely responsible for BFA biosynthesisfrom Eupenicillium brefeldianum ATCC 58665. We furtherdemonstrate that using a product-based assay, important programmingrules of HRPKSs were elucidated, including NADPH concentration-dependentextent of reduction and the control of HRPKS product chain lengthby an associated discrete thiohydrolase (TH). These insights furtherimprove our understanding of how HRPKSs function.


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

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

Putative biosynthethicpathway for 1. The HRPKS isproposed to synthesize the precisely reduced octaketide precursor,which could then be directly offloaded by the thiohydrolase enzymefollowed by a P450-mediated formation of the cyclopentane ring andmacrocyclization to afford the 7-deoxy BFA 2 (top scheme).Alternatively, the first ring annulation can also occur on the ACP-tetheredintermediate before the thiohydrolase release and lactonization (bottomscheme). The C7-hydroxylation is believed to be the final step inthe process to obtain the final structure of 1.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Putative biosynthethicpathway for 1. The HRPKS isproposed to synthesize the precisely reduced octaketide precursor,which could then be directly offloaded by the thiohydrolase enzymefollowed by a P450-mediated formation of the cyclopentane ring andmacrocyclization to afford the 7-deoxy BFA 2 (top scheme).Alternatively, the first ring annulation can also occur on the ACP-tetheredintermediate before the thiohydrolase release and lactonization (bottomscheme). The C7-hydroxylation is believed to be the final step inthe process to obtain the final structure of 1.
Mentions: In this study, we chose the HRPKS responsiblefor brefeldin A (BFA)as a model system (Figure 1). BFA (1) is a protein-transport inhibitor isolated from several speciesof filamentous fungi.10−14 It is used to study protein transport among eukaryotes but has alsobeen found to have antiviral, antifungal, and antitumor properties.15 The polyketide origin of this 16-membered macrolactonewas previously established through feeding studies with labeled acetate.16−18 The proposed biosynthesis of BFA involves formation of an acyclicpolyketide chain that is differentially tailored throughout the backbone(Figure 1). The presence of the terminal hydroxylgroup, along with the strategically positioned double bonds, is proposedto enable cyclization of the acyclic precursor into the fused, bicyclicstructure seen in BFA. We hypothesized that a single HRPKS shouldbe sufficient to generate the entire carbon backbone without the needfor an additional PKS. If the acyclic product indeed contains conjugateddouble bonds as proposed, the biosynthetic product should be readilyidentifiable and isolated. Collectively, the BFA HRPKS appears tofit the mold as a good model HR-PKS for biochemical analysis. Here,we first identified the HRPKS most likely responsible for BFA biosynthesisfrom Eupenicillium brefeldianum ATCC 58665. We furtherdemonstrate that using a product-based assay, important programmingrules of HRPKSs were elucidated, including NADPH concentration-dependentextent of reduction and the control of HRPKS product chain lengthby an associated discrete thiohydrolase (TH). These insights furtherimprove our understanding of how HRPKSs function.

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