- Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi
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Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATPindependent fashion. The KU42 TE domain selects cysteine and homocysteine and catalyzes transthioesterification using the side-chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcases the functional diversity of HRPKS enzymes and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.
- Tang, Man-Cheng,Fischer, Curt R.,Chari, Jason V.,Tan, Dan,Suresh, Sundari,Chu, Angela,Miranda, Molly,Smith, Justin,Zhang, Zhuan,Garg, Neil K.,Onge, Robert P. St.,Tang, Yi
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- Enzymatic Reconstitution and Biosynthetic Investigation of the Bacterial Carbazole Neocarazostatin A
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Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.
- Liu, Yating,Su, Li,Fang, Qing,Tabudravu, Jioji,Yang, Xiaohui,Rickaby, Kirstie,Trembleau, Laurent,Kyeremeh, Kwaku,Deng, Zixin,Deng, Hai,Yu, Yi
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- Seven-enzyme in vitro cascade to (3R)-3-hydroxybutyryl-CoA
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Economical and environmentally-friendly routes to convert feedstock chemicals like acetate into valuable chiral products such as (R)-3-hydroxybutyrate are in demand. Here, seven enzymes (CoaA, CoaD, CoaE, ACS, BktB, PhaB, and GDH) are employed in a one-pot, in vitro, biocatalytic synthesis of (3R)-3-hydroxybutyryl-CoA, which was readily isolated. This platform generates not only chiral diketide building blocks but also desirable CoA derivatives.
- Valencia, Luis E.,Zhang, Zhicheng,Cepeda, Alexis J.,Keatinge-Clay, Adrian T.
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supporting information
p. 1375 - 1378
(2019/02/14)
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- PH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism
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FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzes the dehydration of a 3-hydroxybutyryl-SACP to the (E)-3-butenoyl-SACP. The steady-state kinetic parameters, kcat and kcat/Km, were determined over the pH range 3.0 to 9.2 for the FosDH1-catalyzed dehydration of the N-acetycsteamine thioester, 3-hydroxybutyryl-SNAC (3), to (E)-3-butenoyl-SNAC (4). The pH rate profiles for both log(kcat) and log(kcat/Km) each corresponded to a single pH-dependent ionization to give an active site general base, with a calculated pKa 6.1 ± 0.2 for kcat and pKa 5.7 ± 0.1 for kcat/Km. These results are inconsistent with the commonly suggested "two-base" (base-acid) mechanism for the dehydratases of PKS and fatty acid biosynthesis and support a simple one-base mechanism in which the universally conserved active site His residue acts as the base to deprotonate C-2 of the substrate, then redonates the proton to the C-3 hydroxyl group to promote C-O bond-cleavage and elimination of water. The carboxylate of the paired Asp or Glu residue is thought to bind and orient the hydroxyl group of the substrate in the stereoelectonically favored conformation.
- Xie, Xinqiang,Cane, David E.
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p. 9165 - 9170
(2019/01/03)
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- In vitro kinetic study of the squalestatin tetraketide synthase dehydratase reveals the stereochemical course of a fungal highly reducing polyketide synthase
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Six potential diketide substrates for the squalestatin tetraketide synthase (SQTKS) dehydratase (DH) domain were synthesised as N-acetyl cysteamine thiolesters (SNAC) and tested in kinetic assays as substrates with an isolated DH domain. 3R-3-hydroxybutyryl SNAC 3R-16 was turned over by the enzyme, but its enantiomer was not. Of the four 2-methyl substrates only 2R,3R-2-methyl-3-hydroxybutyryl SNAC 2R,3R-8 was a substrate. Combined with stereochemical information from the isolated SQTKS enoyl reductase (ER) domain, our results provide a near complete stereochemical description of the first cycle of beta-modification reactions of a fungal highly reducing polyketide synthase (HR-PKS). The results emphasise the close relationship between fungal HR-PKS and vertebrate fatty acid synthases (vFAS).
- Liddle, Emma,Scott, Alan,Han, Li-Chen,Ivison, David,Simpson, Thomas J.,Willis, Christine L.,Cox, Russell J.
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supporting information
p. 1727 - 1730
(2017/02/10)
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- Enediyne polyketide synthases stereoselectively reduce the β-ketoacyl intermediates to β- D -hydroxyacyl intermediates in enediyne core biosynthesis
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PKSE biosynthesizes an enediyne core precursor from decarboxylative condensation of eight malonyl-CoAs. The KR domain of PKSE is responsible for iterative β-ketoreduction in each round of polyketide chain elongation. KRs from selected PKSEs were investiga
- Ge, Hui-Ming,Huang, Tingting,Rudolf, Jeffrey D.,Lohman, Jeremy R.,Huang, Sheng-Xiong,Guo, Xun,Shen, Ben
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supporting information
p. 3958 - 3961
(2014/08/18)
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