Methymycin Biosynthesis. Isolation of P450 Monooxygenase Activity in a Cell-Free System from Streptomyces venezuelae

Cane, David E.; Graziani, Edmund I.

doi:10.1021/ja974321v

Cited by 16 publications

(8 citation statements)

References 16 publications

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“…Polyketide cytochrome P450 monooxygenases catalyze the site-specific oxidation of the precursors to many macrolide antibiotics, including erythromycin (1-3), tylosin (4), mycinamicin (5), oleandomycin (6), and methymycin (7). These reactions occur in the late stages of biosynthesis after formation of the macrocycle by the polyketide synthase (PKS).…”

mentioning

confidence: 99%

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,

Betlach¹,

Kealey²,

Ashley³

et al. 1998

Biochemistry

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The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-His) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-His was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a kcat of 1.4 s-1, which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.

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mentioning

confidence: 99%

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,

Betlach¹,

Kealey²,

Ashley³

et al. 1998

Biochemistry

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“…An attempt to detect the oxidation of the aglycones using a cell-free extract of S. venezuelae in the presence of appropriate redox cofactors was unsuccessful. [16] This result suggested that the glycosylation of 1 into 2 precedes the hydroxylation by PikC. However, methynolide and neomethynolide, which are the hydroxylated aglycones by PikC before attaching desosamine, were isolated in the mutant strain of S. venezuelae in which one of genes, desI, involved in desosamine biosynthesis was inactivated, indicating that PikC can catalyze the hydroxylation of non-glycosylated aglycones as well.…”

Section: Analysis Of Des Deletion Strain Of S Venezuelae As a Host Fmentioning

confidence: 98%

“…[7] LC/MS (data not shown) and MS/MS analyses of the predicted compounds provided clear information on catalysis of EryF on both 1 and 6 ( Figures 4B and 4C). We detected the parent ions (M þ H þ ) of the expected hydroxylated compounds of 12-membered (15) and 14-membered (16) (Figure 4A) macrolatones at m/z ¼ 313 and 369, respectively. From each MS/MS analysis of 12-and 14-membered macrolactones, two characteristic dehydration products were detected.…”

Section: Oxidation Of 10-deoxymethynolide and Narbonolide By Eryf Andmentioning

confidence: 99%

Structural Diversification of Macrolactones by Substrate-Flexible Cytochrome P450 Monooxygenases

Lee

Basnet

Hong

et al. 2005

Advanced Synthesis & Catalysis

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The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C-4 position, which is different from the intrinsic C-12 hydroxylation position in the natural substrate. This is the first report of C-4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14-membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12-membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post-PKS oxidations.

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“…For eample, we have characterized two cytochrome P450s that modify the originally formed macrolactone, the erythromycin C-12 hydroxylase, EryK (Ralph Lambalot, now at ABBVie Pharmaceuticals) 126 and the analogous PicK hydroxylase of methymycin/pikromycin biosynthesis (Dr Edmund Graziani, now at Pfizer). 127,128 Our studies of PKS enzymology have been enhanced and broadened by our collaboration with Prof Adrian Keatinge-Clay, a crystallographer from the University of Texas, with remarkably deep insights into the relationship between KR stereochemistry and function. Adrian and his students have solved the structures of a wide variety of PKS domains, with particular attention to the protein structural basis of ketoreductase (KR) and dehydratase stereospecificity.…”

Section: Enzymology and Molecular Genetics Of Natural Product Biosyntmentioning

confidence: 99%

Nature as organic chemist

Cane

2016

J Antibiot

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Figure 15 Stereospecific reduction of (2R)-2-methyl-3-ketopentanoyl-ACP by recombinant ketoreductase (KR) domains: EryKR6, from module 6 of the 6dEB synthase (DEBS); TylKR1, from module 1 of the tylactone synthase; EryKR1, from DEBS module 1; RifKR7, from module 7 of the rifamycin PKS. The (2R)-2-methyl-3-ketopentanoyl-ACP substrate is generated in situ by a reconstituted mixture of dissected PKS domains, Ery[KS6][AT6] (ketosynthase-acyl transferase didomain) and EryACP6 are both from DEBS module 6. Editorial

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Methymycin Biosynthesis. Isolation of P450 Monooxygenase Activity in a Cell-Free System from Streptomyces venezuelae

Cited by 16 publications

References 16 publications

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,

Structural Diversification of Macrolactones by Substrate-Flexible Cytochrome P450 Monooxygenases

Nature as organic chemist

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Methymycin Biosynthesis. Isolation of P450 Monooxygenase Activity in a Cell-Free System from Streptomyces venezuelae

Cited by 16 publications

References 16 publications

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin,

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin,

Structural Diversification of Macrolactones by Substrate-Flexible Cytochrome P450 Monooxygenases

Nature as organic chemist

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Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,

Characterization of the Macrolide P-450 Hydroxylase from Streptomyces venezuelae Which Converts Narbomycin to Picromycin^,