Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ACCESS DATA

Json Xml

Publications

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.

| S-EPMC1857356 | biostudies-literature

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.

| S-EPMC3010413 | biostudies-literature

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

Project description:Streptomyces avermitilis, an industrial organism responsible for the production of the anthelminthic avermectins, harbors a 13.4 kb gene cluster containing 13 unidirectionally transcribed open reading frames corresponding to the apparent biosynthetic operon for the sesquiterpene antibiotic pentalenolactone. The advanced intermediate pentalenolactone F, along with the shunt metabolite pentalenic acid, could be isolated from cultures of S. avermitilis, thereby establishing that the pentalenolactone biosynthetic pathway is functional in S. avermitilis. Deletion of the entire 13.4 kb cluster from S. avermitilis abolished formation of pentalenolactone metabolites, while transfer of the intact cluster to the pentalenolactone nonproducer Streptomyces lividans 1326 resulted in production of pentalenic acid. Direct evidence for the biochemical function of the individual biosynthetic genes came from expression of the ptlA gene (SAV2998) in Escherichia coli. Assay of the resultant protein established that PtlA is a pentalenene synthase, catalyzing the cyclization of farnesyl diphosphate to pentalenene, the parent hydrocarbon of the pentalenolactone family of metabolites. The most upstream gene in the cluster, gap1 (SAV2990), was shown to correspond to the pentalenolactone resistance gene, based on expression in E. coli and demonstration that the resulting glyceraldehyde-3-phosphate dehydrogenase, the normal target of pentalenolactone, was insensitive to the antibiotic. Furthermore, a second GAPDH isozyme (gap2, SAV6296) has been expressed in E. coli and shown to be inactivated by pentalenolactone.

| S-EPMC2518623 | biostudies-literature

Genome mining in Streptomyces avermitilis: A biochemical Baeyer-Villiger reaction and discovery of a new branch of the pentalenolactone family tree.

Project description:Incubation of 1-deoxy-11-oxopentalenic acid (12) with recombinant PtlE protein from Streptomyces avermitilis in the presence of NADPH and catalytic FAD gave the Baeyer-Villiger oxidation product, the previously unknown compound neopentalenolactone D (13), representing a new branch of the pentalenolactone biosynthetic pathway. The structure and stereochemistry of the derived neopentalenolactone D methyl ester (13-Me) were fully assigned by a combination of GC-MS and NMR analysis and confirmed by X-ray crystallography. Neopentalenolactone D (13) was also isolated from engineered cultures of S. avermitilis from which the ptlD gene within the 13.4-kb (neo)-ptl biosynthetic gene cluster had been deleted. The DeltaptlEDeltaptlD double deletion mutant accumulated 12, the substrate for the ptlE gene product, while the corresponding single DeltaptlE mutant produced 12 as well as the related oxidation products 14 and 15. Engineered strains of S. avermitilis, SUKA5 and pKU462::ermRp-ptl cluster, harboring the complete (neo)ptl cluster produced the oxidized lactone 18 and the closely related seco acid hydrolysis products 16 and 17.

| S-EPMC2747811 | biostudies-other

Genome mining in Streptomyces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone.

Project description:The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and Streptomyces arenae TU?469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from Streptomyces avermitilis, each catalyze the Fe(2+)-?-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ?penD and ?pntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ?ptlE ?ptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ?ptlE ?ptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).

| S-EPMC3051010 | biostudies-literature

Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis.

Project description:Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities.The respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D.We demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential.

| S-EPMC5240415 | biostudies-literature

Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial compound FR900098.

Project description:The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent antibacterial and antimalarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.

| S-EPMC2603629 | biostudies-literature

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.

Project description:To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.

| S-EPMC143579 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data