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Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis.

ABSTRACT: The hydroxylase encoded by the ptlH (SAV2991) gene from the pentalenolactone gene cluster of Streptomyces avermitilis was cloned by PCR and expressed in Escherichia coli as an N-terminal His6-tag protein. Incubation of recombinant PtlH with (+/-)-1-deoxypentalenic acid (5) in the presence of Fe(II), alpha-ketoglutarate, and O2 gave (-)-11beta-hydroxy-1-deoxypentalenic acid (8), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat = 4.2 +/- 0.6 s-1 and Km (5) = 0.57 +/- 0.19 mM. 8 is a new intermediate in the conversion of the sesquiterpene pentalenene (3) to pentalenolactone (1).

SUBMITTER: You Z

PROVIDER: S-EPMC2533727 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis.

You Zheng Z Omura Satoshi S Ikeda Haruo H Cane David E DE

Journal of the American Chemical Society 20060501 20

The hydroxylase encoded by the ptlH (SAV2991) gene from the pentalenolactone gene cluster of Streptomyces avermitilis was cloned by PCR and expressed in Escherichia coli as an N-terminal His6-tag protein. Incubation of recombinant PtlH with (+/-)-1-deoxypentalenic acid (5) in the presence of Fe(II), alpha-ketoglutarate, and O2 gave (-)-11beta-hydroxy-1-deoxypentalenic acid (8), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The s ...[more]

PMID: 16704250

Similar Datasets

Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlI, a cytochrome P450 of Streptomyces avermitilis.

Project description:A gene cluster encoding all of the enzymes for the biosynthesis of the antibiotic pentalenolactone (1) has recently been identified in Streptomyces avermitilis. The biosynthetic gene cluster contains the ptlI (SAV2999) gene which encodes a cytochrome P450 (CYP183A1). PtlI was cloned by PCR and expressed in Escherichia coli as a C-terminal His6-tag protein. Recombinant PtlI bound pentalenene (3) with high affinity (KD = 1.44 +/- 0.14 muM). Incubation of recombinant PtlI with (+/-)-3 in the presence of NADPH, E. coli flavodoxin and flavodoxin reductase, and O2 resulted in conversion to a single enantiomer of pentalen-13-al (7), by stepwise allylic oxidation via pentalen-13-ol (6). The steady-state kinetic parameters for the oxidation of pentalenene (3) to pentalen-13-ol (6) were kcat = 0.503 +/- 0.006 min-1 and Km = 3.33+/-0.62 muM for 3.

| S-EPMC2533730 | biostudies-literature

Pentalenolactone biosynthesis: Molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate.

Project description:Pentalenolactone (1) is an antibiotic that has been isolated from many species of Streptomyces. The putative dehydrogenase encoded by the ptlF gene (SAV2993) found within the Streptomyces avermitilis pentalenolactone gene cluster was cloned and overexpressed in Escherichia coli. PtlF, which belongs to the short-chain dehydrogenase/oxidoreductase superfamily, was shown to catalyze the oxidation of 1-deoxy-11beta-hydroxypentalenic acid (9) to 1-deoxy-11-oxopentalenic acid (10), a new intermediate of the pentalenolactone biosynthetic pathway. The methyl ester of 10 was characterized by NMR, GC-MS and high resolution mass spectrometry. PtlF exhibited a 150-fold preference for beta-NAD(+) over beta-NADP(+). PtlF had a pH optimum of 8.0 in the physiological pH range, while a significant activity enhancement was observed from pH 9.0 to 11.3. At pH 8.0, PtlF had a k(cat) of 0.65+/-0.03 s(-1), with a K(m) for 9 of 6.5+/-1.5 microM and K(m) for NAD(+) of 25+/-3 microM.

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Crystal structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis.

Project description:The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal alpha-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.

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A gene cluster for biosynthesis of the sesquiterpenoid antibiotic pentalenolactone in Streptomyces avermitilis.

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SAV4189, a MarR-Family Regulator in <i>Streptomyces avermitilis</i>, Activates Avermectin Biosynthesis.

Project description:The bacterial species Streptomyces avermitilis is an important industrial producer of avermectins, which are widely utilized as effective anthelmintic and insecticidal drugs. We used gene deletion, complementation, and overexpression experiments to identify SAV4189, a MarR-family transcriptional regulator (MFR) in this species, as an activator of avermectin biosynthesis. SAV4189 indirectly stimulated avermectin production by altering expression of cluster-situated activator gene aveR, and directly repressed the transcription of its own gene (sav_4189) and adjacent cotranscribed gene sav_4190 (which encodes an unknown transmembrane efflux protein). A consensus 13-bp palindromic sequence, 5'-TTGCCYKHRSCAA-3' (Y = T/C; K = T/G; H = A/C/T; R = A/G; S = C/G), was found within the SAV4189-binding sites of its own promoter region, and shown to be essential for binding. The SAV4189 regulon was thus predicted based on bioinformatic analysis. Night new identified SAV4189 targets are involved in transcriptional regulation, primary metabolism, secondary metabolism, and stress response, reflecting a pleiotropic role of SAV4189. sav_4190, the important target gene of SAV4189, exerted a negative effect on avermectin production. sav_4189 overexpression and sav_4190 deletion in S. avermitilis wild-type and industrial strains significantly increased avermectin production. SAV4189 homologs are widespread in other Streptomyces species. sav_4189 overexpression in the model species S. coelicolor also enhanced antibiotic production. The strategy of increasing yield of important antibiotics by engineering of SAV4189 homologs and target gene may potentially be extended to other industrial Streptomyces species. In addition, SAV4189 bound and responded to exogenous antibiotics hygromycin B and thiostrepton to modulate its DNA-binding activity and transcription of target genes. SAV4189 is the first reported exogenous antibiotic receptor among Streptomyces MFRs.

| S-EPMC6036246 | biostudies-literature

Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains.

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