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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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Summary ofthe programming rules exhibited by Bref-PKS. From thein vitro studies, we were able to fully reconstitute the complex programmingof this model HRPKS. Bref-PKS uses different permutations of the reductivedomains at each extension cycle and selectively offloads the correctoctaketide products with the partner Bref-TH or the longer nonaketideproducts with base hydrolysis. Compounds 4 and 6 that resulted from additional enoyl reduction at the finalextension are italicized.
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fig6: Summary ofthe programming rules exhibited by Bref-PKS. From thein vitro studies, we were able to fully reconstitute the complex programmingof this model HRPKS. Bref-PKS uses different permutations of the reductivedomains at each extension cycle and selectively offloads the correctoctaketide products with the partner Bref-TH or the longer nonaketideproducts with base hydrolysis. Compounds 4 and 6 that resulted from additional enoyl reduction at the finalextension are italicized.

Mentions: Using theBref-PKS and Bref-TH pair (as well as the Fma pair),we showed that the releasing enzyme is important in ensuring the properchain length control of HRPKSs. In the absence of the TH enzymes,both Bref-PKS and Fma-PKS synthesized longer products than what isreflected in the final product. This result can be rationalized ina competition model in which the polyketide chain can be either offloadedby the TH/AT at the correct size or can be recaptured by the KS domainfor another round of elongation (Figure 6).In the absence of the TH, the polyketide chain remains attached tothe ACP domain, which allows reentry into the KS domain. If the KScan accommodate a product of longer size, an additional extensionstep can take place as observed in products 7 and 10. We did not detect any shorter polyketides in the in vitroassays, indicating the high substrate specificities of the TH towardthe correct acyl group. However, when equimolar amount of Bref-THwas added 16 h after initiation of the in vitro Bref-PKS reaction,the longer nonaketide products (7 and 8)were again observed at similar levels as the base-hydrolyzed reaction(Supplementary Figure S10). This indicatesthat Bref-TH is capable of hydrolyzing longer chain length but preferablyhydrolyzes the correct octaketide chain in a timely manner when co-incubatedwith Bref-PKS. Interestingly, in both Bref-PKS and Fma-PKS, the extraketide(s) that form as a result of excluding the TH in the reactionwere completely processed by the available reduction domains. Therecognition of the longer (and unnatural) substrates by these domainsmay similarly be due to stalling of the polyketide on the ACP domain,which led to the observed modification. Detailed kinetics studiesusing model substrates of varying length will provide insights intothe substrate specificities of these tailoring domains.


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

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

Summary ofthe programming rules exhibited by Bref-PKS. From thein vitro studies, we were able to fully reconstitute the complex programmingof this model HRPKS. Bref-PKS uses different permutations of the reductivedomains at each extension cycle and selectively offloads the correctoctaketide products with the partner Bref-TH or the longer nonaketideproducts with base hydrolysis. Compounds 4 and 6 that resulted from additional enoyl reduction at the finalextension are italicized.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Summary ofthe programming rules exhibited by Bref-PKS. From thein vitro studies, we were able to fully reconstitute the complex programmingof this model HRPKS. Bref-PKS uses different permutations of the reductivedomains at each extension cycle and selectively offloads the correctoctaketide products with the partner Bref-TH or the longer nonaketideproducts with base hydrolysis. Compounds 4 and 6 that resulted from additional enoyl reduction at the finalextension are italicized.
Mentions: Using theBref-PKS and Bref-TH pair (as well as the Fma pair),we showed that the releasing enzyme is important in ensuring the properchain length control of HRPKSs. In the absence of the TH enzymes,both Bref-PKS and Fma-PKS synthesized longer products than what isreflected in the final product. This result can be rationalized ina competition model in which the polyketide chain can be either offloadedby the TH/AT at the correct size or can be recaptured by the KS domainfor another round of elongation (Figure 6).In the absence of the TH, the polyketide chain remains attached tothe ACP domain, which allows reentry into the KS domain. If the KScan accommodate a product of longer size, an additional extensionstep can take place as observed in products 7 and 10. We did not detect any shorter polyketides in the in vitroassays, indicating the high substrate specificities of the TH towardthe correct acyl group. However, when equimolar amount of Bref-THwas added 16 h after initiation of the in vitro Bref-PKS reaction,the longer nonaketide products (7 and 8)were again observed at similar levels as the base-hydrolyzed reaction(Supplementary Figure S10). This indicatesthat Bref-TH is capable of hydrolyzing longer chain length but preferablyhydrolyzes the correct octaketide chain in a timely manner when co-incubatedwith Bref-PKS. Interestingly, in both Bref-PKS and Fma-PKS, the extraketide(s) that form as a result of excluding the TH in the reactionwere completely processed by the available reduction domains. Therecognition of the longer (and unnatural) substrates by these domainsmay similarly be due to stalling of the polyketide on the ACP domain,which led to the observed modification. Detailed kinetics studiesusing model substrates of varying length will provide insights intothe substrate specificities of these tailoring domains.

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