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6-deoxyerythronolide B synthase

Xinqiang Xie, Ashish Garg, Chaitan Khosla, David E Cane
The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0 ) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product...
March 1, 2017: Journal of the American Chemical Society
Irina Koryakina, Christian Kasey, John B McArthur, Andrew N Lowell, Joseph A Chemler, Shasha Li, Douglas A Hansen, David H Sherman, Gavin J Williams
Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another...
January 20, 2017: ACS Chemical Biology
Omer Ad, Benjamin W Thuronyi, Michelle C Y Chang
Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C-C bond formation, and chain transfer, to the overall selectivity and efficiency of this process...
January 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
Thomas Robbins, Joshuah Kapilivsky, David E Cane, Chaitan Khosla
Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase...
August 16, 2016: Biochemistry
Matthew P Ostrowski, David E Cane, Chaitan Khosla
Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS)...
July 2016: Journal of Antibiotics
Panos Argyropoulos, Fabien Bergeret, Christophe Pardin, Janice M Reimer, Atahualpa Pinto, Christopher N Boddy, T Martin Schmeing
Type I polyketide synthases (PKSs) are giant multidomain proteins that synthesize many therapeutics and other natural products. The synthesis proceeds by a thiotemplate mechanism whereby intermediates are covalently attached to the PKS. The release of the final polyketide is catalyzed by the terminal thioesterase (TE) domain through hydrolysis, transesterification, or macrocyclization. The PKS 6-deoxyerythronolide B synthase (DEBS) produces the 14-membered macrolide core of the clinically important antibiotic erythromycin...
March 2016: Biochimica et Biophysica Acta
Matthew N R Johnson, Casey H Londergan, Louise K Charkoudian
Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4'-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP-enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis...
August 13, 2014: Journal of the American Chemical Society
Ashish Garg, Xinqiang Xie, Adrian Keatinge-Clay, Chaitan Khosla, David E Cane
Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4)...
July 23, 2014: Journal of the American Chemical Society
Briana J Dunn, Katharine R Watts, Thomas Robbins, David E Cane, Chaitan Khosla
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS)...
June 17, 2014: Biochemistry
Chaitan Khosla, Daniel Herschlag, David E Cane, Christopher T Walsh
Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions...
May 13, 2014: Biochemistry
Andrea L Edwards, Tsutomu Matsui, Thomas M Weiss, Chaitan Khosla
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification...
May 29, 2014: Journal of Molecular Biology
Brian Lowry, Thomas Robbins, Chih-Hisang Weng, Robert V O'Brien, David E Cane, Chaitan Khosla
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min(-1), comparable to the turnover rate of a truncated trimodular derivative (2...
November 13, 2013: Journal of the American Chemical Society
Satoshi Yuzawa, Clara H Eng, Leonard Katz, Jay D Keasling
LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in Streptomyces aureofaciens Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA...
June 4, 2013: Biochemistry
John Yan, Christopher Hazzard, Shilah A Bonnett, Kevin A Reynolds
The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products...
November 20, 2012: Biochemistry
Atahualpa Pinto, Meng Wang, Mark Horsman, Christopher N Boddy
Macrocyclic polyketide natural products are an indispensable source of therapeutic agents. The final stage of their biosynthesis, macrocyclization, is catalyzed regio- and stereoselectively by a thioesterase. A panel of substrates were synthesized to test their specificity for macrocyclization by the erythromycin polyketide synthase TE (DEBS TE) in vitro. It was shown that DEBS TE is highly stereospecific, successfully macrocyclizing a 14-member ring substrate with an R configured O-nucleophile, and highly regioselective, generating exclusively the 14-member lactone over the 12-member lactone...
May 4, 2012: Organic Letters
Satoshi Yuzawa, Shiven Kapur, David E Cane, Chaitan Khosla
The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg...
May 8, 2012: Biochemistry
Shiven Kapur, Brian Lowry, Satoshi Yuzawa, Sanketha Kenthirapalan, Alice Y Chen, David E Cane, Chaitan Khosla
Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase...
March 13, 2012: Proceedings of the National Academy of Sciences of the United States of America
Jianting Zheng, Adrian T Keatinge-Clay
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...
July 1, 2011: Journal of Molecular Biology
Louise K Charkoudian, Corey W Liu, Stefania Capone, Shiven Kapur, David E Cane, Antonio Togni, Dieter Seebach, Chaitan Khosla
The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain...
July 2011: Protein Science: a Publication of the Protein Society
Amanda J Hughes, Adrian Keatinge-Clay
In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS studies...
February 25, 2011: Chemistry & Biology
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