Publications

• Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases.

  Zheng J., Keatinge-Clay A.T.

  The process by which α-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase h ... >> More

  The process by which α-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase has been proposed to operate as a racemase, aiding in the epimerization process that reverses the orientation of the α-methyl group of the polyketide intermediate generated by the ketosynthase to the configuration observed in the 6-deoxyerythronolide B final product. To learn more about the epimerization process, the structure of the C2-type KR from the third module of the pikromycin synthase, analogous to the KR from the third module of 6-deoxyerythronolide B synthase, was determined to 1.88 Å resolution. This first structural analysis of this KR-type reveals differences from reductase-competent KRs such as that the site NADPH binds to reductase-competent KRs is occluded by side chains and the putative catalytic tyrosine possesses more degrees of freedom. The active-site geometry may enable C2-type KRs to align the thioester and β-keto groups of a polyketide intermediate to reduce the pK(a) of the α-proton and accelerate its abstraction. Results from in vivo assays of engineered PKSs support that C2-type KRs cooperate with epimer-specific ketosynthases to set the configurations of substituent-bearing α-carbons. << Less

  J. Mol. Biol. 410:105-117(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase.

  Lu H., Tsai S.C., Khosla C., Cane D.E.

  The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a ... >> More

  The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct. << Less

  Biochemistry 41:12590-12597(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural rearrangements of a polyketide synthase module during its catalytic cycle.

  Whicher J.R., Dutta S., Hansen D.A., Hale W.A., Chemler J.A., Dosey A.M., Narayan A.R., Haakansson K., Sherman D.H., Smith J.L., Skiniotis G.

  The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS mo ... >> More

  The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules. << Less

  Nature 510:560-564(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.

  Yan J., Gupta S., Sherman D.H., Reynolds K.A.

  The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extens ... >> More

  The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products. << Less

  ChemBioChem 10:1537-1543(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase.

  Yin Y., Lu H., Khosla C., Cane D.E.

  Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each ... >> More

  Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS). << Less

  J. Am. Chem. Soc. 125:5671-5676(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase.

  Tang L., Fu H., Betlach M.C., McDaniel R.

  <h4>Background</h4>A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression sy ... >> More

  <h4>Background</h4>A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates.<h4>Results</h4>The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones.<h4>Conclusions</h4>These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain. << Less

  Chem. Biol. 6:553-558(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Interrogating the molecular basis for multiple macrolactone ring formation by the pikromycin polyketide synthase.

  Kittendorf J.D., Beck B.J., Buchholz T.J., Seufert W., Sherman D.H.

  The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expandin ... >> More

  The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expanding chemical diversity. Here, we describe a series of experiments designed to probe the importance of the protein-protein interaction that occurs between the final two monomodules, PikAIII (module 5) and PikAIV (module 6), for the production of the 12 membered ring macrolactone 10-deoxymethynolide. The results obtained from these in vitro studies demonstrate that PikAIII and PikAIV generate the 12 membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. Accordingly, the data are consistent with PikAIV adopting an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization. << Less

  Chem. Biol. 14:944-954(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.