A Biosynthetic Pathway for BE-7585A, a 2-Thiosugar-Containing Angucycline-Type Natural Product

Sasaki, Eita; Ogasawara, Yasushi; Liu, Hung Wen

doi:10.1021/ja1014037

Cited by 63 publications

(104 citation statements)

References 95 publications

(141 reference statements)

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“…A pET-28b(+) (Novagen)-derived DNA plasmid encoding N-term His-tagged BexL (BexL/pET28b) was prepared from cosmid C006 previously constructed from the genomic DNA of Amycolatopsis orientalis subsp. Vinearia (24). The BexL/pET28b plasmid was transformed into Escherichia coli BL21(DE3)-competent cells and plated on LB-agar plates containing kanamycin (50 μg/mL).…”

Section: Methodsmentioning

confidence: 99%

“…However, many type II PKSs contain di-domain ARO/CYCs that have two seemingly identical domains (18-23). Why these enzymes require two domains (as opposed to just one) and how they conduct the cyclization/aromatization are not understood.The di-domain ARO/CYCs are found in both nonreducing and reducing PKSs (18,24). In nonreducing systems, the didomain ARO/CYCs regiospecifically cyclize a polyketide between C7 and C12, followed by aromatization (18).…”

mentioning

confidence: 99%

“…The di-domain ARO/CYCs are found in both nonreducing and reducing PKSs (18,24). In nonreducing systems, the didomain ARO/CYCs regiospecifically cyclize a polyketide between C7 and C12, followed by aromatization (18).…”

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confidence: 99%

“…The linear poly-β-ketone precursor of BE-7585A is first cyclized, then reduced at the C9 position by KR, and then aromatized by BexL ( Fig. 1) (24). Both StfQ and BexL use 20-carbon poly-β-ketone substrates; therefore, the structural enzymology of these two ARO/CYCs offers a great opportunity for side-by-side comparison with insight into the catalytic mechanisms of di-domain ARO/CYCs.…”

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confidence: 99%

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Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

Caldara-Festin

Jackson

Barajas

et al. 2015

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

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Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-β-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono-and didomain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.polyketide biosynthesis | structural biology | aromatase/cyclase T he biosynthesis of type II aromatic polyketide natural products has been extensively investigated because of the versatile pharmacological properties of these compounds (1-7). The type II polyketide synthase (PKS) is composed of dissociated enzymes that are used iteratively and are responsible for the elongation, cyclization, and modification of the polyketide chain ( Fig. 1) (3,4,8,9). The regiospecific cyclization of an acyl carrier protein (ACP)-linked linear poly-β-ketone intermediate is a key transformation catalyzed by type II PKSs. However, the enzymatic mechanism of cyclization remains poorly understood (10-14). Without such knowledge, the polyketide cyclization pattern cannot be predicted; a full understanding of this process at the molecular level is essential for future biosynthetic engineering efforts.In 2008, we reported the crystal structure of the first aromatase/cyclase (ARO/CYC) (TcmN ARO/CYC), which is a singledomain protein (15). On the basis of the structural analysis and mutagenesis results, we proposed that monodomain ARO/CYCs contain an active site and are capable of catalyzing polyketide cyclization and aromatization. Since then, we have performed structural and biochemical studies of two other monodomain ARO/CYCs: WhiE and ZhuI (Fig. 1) (16, 17). These studies provided strong evidence supporting our hypothesis that ARO/ CYC is the site of polyketide cyclization. However, many type II PKSs contain di-domain ARO/CYCs that have two seemingly identical domains (18-23). Why these enzymes require two domains (as opposed to just one) and how t...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

Caldara-Festin

Jackson

Barajas

et al. 2015

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

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“…612,613 As suggested in Scheme 142, the initial oxidative tetracyclic metabolite 1003 derived from the released product 1002 from the PKS enzyme undergoes oxygenative Baeyer-Villiger type expansion of the D-ring ketone to the lactone 1004 , catalyzed by BexE. 614 Hydrolysis to the hydroxy acid 1005 allows rotation and reformation of an angular ketone 1008 , thereby altering the connectivity from the linear tetracyclic starting substrate. A second Baeyer-Villiger insertion by the BexM enzyme to afford 1009 is proposed to be followed by a ring contraction back to the indicated hydroxy cyclohexanone 1010 .…”

Section: Oxidative Ring Rearrangementmentioning

confidence: 99%

Oxidative Cyclization in Natural Product Biosynthesis

et al. 2016

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Oxidative cyclizations are important transformations that occur widely during natural product biosynthesis. The transformations from acyclic precursors to cyclized products can afford morphed scaffolds, structural rigidity and biological activities. Some of the most dramatic structural alterations in natural product biosynthesis occur through oxidative cyclization. In this review, we examine the different strategies used by Nature to create new intra-(inter-)-molecular bonds via redox chemistry. The review will cover both oxidation- and reduction-enabled cyclization mechanisms, with an emphasis on the former. Radical cyclizations catalyzed by P450, nonheme iron, α-KG dependent oxygenases and radical SAM enzymes are discussed to illustrate the use of molecular oxygen and S-adenosylmethionine to forge new bonds at unactivated sites via one-electron manifolds. Nonradical cyclizations catalyzed by flavin-dependent monooxygenases and NAD(P)H-dependent reductases are covered to show the use of two-electron manifolds in initiating cyclization reactions. The oxidative installation of epoxides and halogens into acyclic scaffolds to drive subsequent cyclizations are separately discussed as examples of “disappearing” reactive handles. Lastly, oxidative rearrangement of rings systems, including contractions and expansions will be covered.

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