The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units

Wu, Kai; Chung, Loleta; Revill, W. Peter; Katz, Leonard; Reeves, Christopher D.

doi:10.1016/s0378-1119(00)00171-2

Cited by 254 publications

(259 citation statements)

References 25 publications

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“…Genes for FkbO 520 (28) and Hyg5 (42) were amplified by PCR from genomic DNA isolated from S. hygroscopicus var. ascomyceticus and S. hygroscopicus, respectively.…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Andexer

Kendrew²,

Nur‐e‐Alam³

et al. 2011

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

101

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The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4 R ,5 R )-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus . Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

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“…Genes for FkbO 520 (28) and Hyg5 (42) were amplified by PCR from genomic DNA isolated from S. hygroscopicus var. ascomyceticus and S. hygroscopicus, respectively.…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Andexer

Kendrew²,

Nur‐e‐Alam³

et al. 2011

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

101

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“…The C-2 hydroxy group of this extender unit is usually but not always (65) methylated. A subcluster of five genes, asm13-17, which form an operon, is evidently responsible for the formation of the substrate for this particular chain-extension step, as has been proposed earlier for the homologous genes fkbG-K in the FK520 cluster (46). In recent work, coexpression in Streptomyces lividans of asm13-17 with a cassette of eryABC, encoding the erythromycin PKS, in which the AT6 had been replaced with the hydroxymalonate-specifying AT8 from the FK520 PKS, resulted in the formation of 2-desmethyl-2-methoxy-6-deoxyerythronolide B by incorporation of a methoxymalonate instead of a methylmalonate unit, whereas asm13-16 in place of asm13-17 gave no new product (66).…”

Section: Discussionmentioning

confidence: 57%

“…asm17 is probably cotranscribed with asm13-16 and encodes an Omethyltransferase. The biosynthetic gene cluster for FK520, a macrolide also involving ''glycolate'' extender units, contains a set of five genes, fkbGHIJK, which are homologous to asm13-17 (46). This fact further supports the notion that asm13-17 are involved in the synthesis of this unusual polyketide chain extension unit.…”

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 57%

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

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

279

256

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Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual ''methoxymalonate'' polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

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“…Meridamycin is a 27-membered macrocyclic polyketide, isolated independently from two different streptomycete strains (Fehr et al, 1994;Salituro et al, 1995). Although it lacks immunosuppressant activity, it shares the structural motif common to the macrocyclic polyketide immunosuppressants rapamycin (Schwecke et al, 1995), FK506 (Motamedi & Shafiee, 1998) and FK520 (Wu et al, 2000), which is known to interact with the hydrophobic cavity on the surface of FK506-binding protein-12 (FKBP-12) (Van Duyne et al, 1991, 1993. This structural moiety is proposed to mimic a natural peptide substrate and hence inhibit the peptidyl-prolyl isomerase activity of FKBP-12.…”

Section: Introductionmentioning

confidence: 99%

Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin

et al. 2006

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Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.

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The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units

Cited by 254 publications

References 25 publications

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin

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