Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits

McDaniel, Robert; Ebert-Khosla, Susanne; Hopwood, David A.; Khosla, Chaitan

doi:10.1038/375549a0

Cited by 276 publications

(209 citation statements)

References 30 publications

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“…However, we cannot rule out the possibility that JadG can catalyze C-12 monooxygenation because the monooxygenation can take place spontaneously after 4a,12b-dehydration (8), here catalyzed by JadH. Similar spontaneous monooxygenations were also observed in shunt products (aloesaponarin II and 3,8-dihydroxy-1-ethylanthraquinone-2-carboxylic acid) of actinorhodin, whose quinone moiety was actually formed by the JadG homologue, ActVA-Orf6 (38,39).…”

Section: Discussionmentioning

confidence: 75%

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

Chen

Wang

Greenwell

et al. 2005

Journal of Biological Chemistry

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A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase ␣ (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the postpolyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

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Section: Discussionmentioning

confidence: 75%

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

Chen

Wang

Greenwell

et al. 2005

Journal of Biological Chemistry

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“…Despite the importance of the polyketide aromatic rings, little is known about how the cyclizations are precisely controlled. Although the inclusion of Tcm ARO/CYC promotes C9-C14 cyclized products in vitro and in vivo (3,4), studies on other ARO/CYC domains have been inconclusive (1): It is never clear whether the polyketide chain is ever bound by ARO/CYC or whether this domain is only an auxiliary protein that promotes proteinprotein interactions. In this work, the 1.9 Å crystal structure of Tcm ARO/CYC reveals that the ARO/CYC has an interior pocket capable of binding a 20-carbon polyketide substrate.…”

Section: Strongly Diminished Rm80 Production (Ratio Of [4 ϩ 5]/[6] ͻmentioning

confidence: 99%

Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides

Ames

Korman

Zhang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

154

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Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure. The 1.9 Å crystal structure of Tcm ARO/CYC reveals that the enzyme belongs to the Bet v1-like superfamily (or STAR domain family) with a helix–grip fold, and contains a highly conserved interior pocket. Docking, mutagenesis, and an in vivo assay show that the size, shape, and composition of the pocket are important to orient and specifically fold the polyketide chain for C9-C14 first-ring and C7-C16 second-ring cyclizations. Two pocket residues, R69 and Y35, were found to be essential for promoting first- and second-ring cyclization specificity. Different pocket residue mutations affected the polyketide product distribution. A mechanism is proposed based on the structure-mutation-docking results. These results strongly suggest that the regiospecific cyclizations of the first two rings and subsequent aromatizations take place in the interior pocket. The chemical insights gleaned from this work pave the foundation toward defining the molecular rules for the ARO/CYC cyclization specificity, whose rational control will be important for future endeavors in the engineered biosynthesis of novel anticancer and antibiotic aromatic polyketides.

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“…Consequently, there has been considerable research into the biosynthesis of these compounds in the hope that by combining a functional understanding of the enzymes of these pathways with the techniques of combinatorial biosynthesis, multiple new drug candidates can be isolated (Hutchinson, 1999). The potential of this methodology is illustrated by numerous reports in which genetically engineered Streptomyces strains have been made to produce novel natural products whose structures have been rationally designed (McDaniel et al, 1995). This work has been aided by the cloning, sequence analysis and manipulation of the gene clusters responsible for the biosynthesis of aromatic and macrolide polyketides and has focused on the enzymes that catalyse the assembly and subsequent cyclization of the polyketide chain (for reviews, see Hopwood & Sherman, 1990;Hutchinson & Fujii, 1995;Hopwood, 1997;Hutchinson, 1997).…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction studies of a monooxygenase fromStreptomyces coelicolorA3(2) involved in the biosynthesis of the polyketide actinorhodin

Kendrew¹,

Federici²,

Savino³

et al. 2000

Acta Crystallogr D Biol Cryst

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The aromatic monooxygenase ActVA-Orf6 from Streptomyces coelicolor A3(2) that catalyses an unusual oxidation on the actinorhodin biosynthetic pathway has been crystallized. The crystals diffract to 1.73 A Ê and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.95, b = 59.29, c = 71.67 A Ê . Solvent-content (44%) and self-rotation function calculations predict the presence of two molecules in the asymmetric unit. Structure determination should provide further insight into the enzyme mechanism and aid in the design of biosynthetic pathways to produce new polyketide natural products with novel functionality.

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Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits

Cited by 276 publications

References 30 publications

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides

Crystallization and preliminary X-ray diffraction studies of a monooxygenase fromStreptomyces coelicolorA3(2) involved in the biosynthesis of the polyketide actinorhodin

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