Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium

Samson, Suellen M.; Belagaje, Rama M.; Blankenship, Dale T.; Chapman, Jerry L.; Perry, David K.; Skatrud, Paul L.; VanFrank, R. M.; Abraham, E. P.; Baldwin, Jack E.; Queener, Stephen W.; Ingolia, Thomas D.

doi:10.1038/318191a0

Cited by 216 publications

(87 citation statements)

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“…17 Isopenicillin N synthase (IPNS) is one of the more well-understood mononuclear non-heme-iron enzyme due to the extensive studies by Baldwin and co-workers. IPNS catalyzes the four electron oxidation of δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) tripeptide to isopenicillin N. 51 Elegant biochemical and crystallographic studies provided convincing evidence that the reaction proceeds via two successive two-electron oxidations (Scheme 6). 7,52 The first oxidation is thought to involve cleavage of the C Cys,β -H bond by a formally Fe(III)-superoxo intermediate, followed by formation of the β-lactam ring, possibly by attack of the valine amidate on the thioaldehyde.…”

Section: Mechanistic Diversity Of Presumptive Fe(iv)-oxo Intermediatementioning

confidence: 99%

Non-Heme Fe(IV)–Oxo Intermediates

et al. 2007

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High-valent non-heme iron-oxo intermediates have been proposed for decades as the key intermediates in numerous biological oxidation reactions. In the last three years, the first direct characterization of such intermediates has been provided by studies of several αKG-dependent oxygenases that catalyze either hydroxylation or halogenation of their substrates. In each case, the Fe(IV)-oxo intermediate is implicated in cleavage of the aliphatic C-H bond to initiate hydroxylation or halogenation. The observation of non-heme Fe(IV)-oxo intermediates and Fe(II)-containing product(s) complexes with almost identical spectroscopic parameters in the reactions of two distantly related αKG-dependent hydroxylases suggests that members of this sub-family follow a conserved mechanism for substrate hydroxylation. In contrast, for the αKG-dependent non-heme-iron halogenase, CytC3, two distinct Fe(IV) complexes form and decay together, suggesting that they are in rapid equilibrium. The existence of two distinct conformers of the Fe site may be the key factor accounting for the divergence of the halogenase reaction from the more usual hydroxylation pathway after C-H cleavage. Distinct transformations catalyzed by other mononuclear non-heme enzymes are likely also to involve initial C-H-cleavage by Fe(IV)-oxo complexes, followed by diverging reactivities of the resulting Fe(III)-hydroxo/substrate radical intermediates.

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Section: Mechanistic Diversity Of Presumptive Fe(iv)-oxo Intermediatementioning

confidence: 99%

Non-Heme Fe(IV)–Oxo Intermediates

et al. 2007

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“…E. coli strains were grown in TY broth (19) supplemented with tetracycline (5 pug/ml) or ampicillin (80 ,ug/ml). DNA techniques followed standard procedures (15).…”

Section: Methodsmentioning

confidence: 99%

“…The gene encoding IPNS, pcbC, was cloned first from C. acremonium (19) and then from Penicillium chrysogenum (3), the producer of penicillins G and V. The deduced amino acid sequences (3,19) provided important new information because direct biochemical determinations were not available from limited amounts of homogeneous IPNS (10,16). The high yield of IPNS from expression of these cloned genes in Escherichia coli (3,19) provided ready access to material for physical and chemical characterizations. Availability of the pcbC gene for in vitro mutagenesis allowed comparison of natural and altered IPNS molecules and resultant independent assessment of the role in catalysis for each of two cysteine residues in C. acremonium IPNS (20).…”

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Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

et al. 1989

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Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.

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“…The recent cloning of antibiotic biosynthesis genes from the gram-positive bacteria of the genera Streptomyces (4,6,7,16,17) and Bacillus (13,20) and also from the filamentous fungi (3,24,25) facilitated studies on the structure, organization, and regulation of these genes at the molecular level (10,21,26). Gramicidin S is a cyclic decapeptide antibiotic produced by Bacillus brevis in the transition phase from vegetative to stationary growth (19).…”

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Organization of the biosynthesis genes for the peptide antibiotic gramicidin S

Krause

Marahiel

1988

J Bacteriol

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A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S.synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.The recent cloning of antibiotic biosynthesis genes from the gram-positive bacteria of the genera Streptomyces (4,6,7,16,17) and Bacillus (13,20) and also from the filamentous fungi (3, 24, 25) facilitated studies on the structure, organization, and regulation of these genes at the molecular level (10,21,26). Gramicidin S is a cyclic decapeptide antibiotic produced by Bacillus brevis in the transition phase from vegetative to stationary growth (19). Gramicidin S biosynthesis is one of the best understood biochemical pathways for a secondary metabolite in Bacillus spp. (11, 12) Two multifunctional enzymes, gramicidin S synthetases 1 and 2, with molecular masses of 120 and 280 kilodaltons (kDa), respectively, are involved in the activation, racemization, and condensation of the constituent amino acids via a thiotemplate mechanism (15) to produce the intact peptide antibiotic (Fig. 1). To understand the organization and regulation of the genes involved in the production of peptide antibiotics in Bacillus spp., we recently cloned the entire tyrocidine synthetase 1 gene, tycA, and studied its regulation in the tyrocidine producer B. brevis ATCC 8185 and in the heterologous host Bacillus subtilis (20,21). We also cloned a 3.9-kilobase (kb) DNA fragment of the gramicidin S synthetase 2 gene; grsB, which directed the synthesis of a 155-kDa ornithine-activating domain (13) (18), was used (Stratagene, San Diego, Calif.). E. coli BHB2688 and BHB2690 were used for the preparation of in vitro packaging extracts and were grown in NZ medium (9,18). Gramicidin S producer B. brevis ATCC 9999 was grown in nutrient broth as described previously (19).Protein crude extracts were prepared. from EMBL315-infected E. coli C600 cells (16) grown at.37°C in NZ medium. Cells were collected before complete lysis, usually at an optical density at 600 nm of about 0.8. Preparation of protein extracts and fractionation with 50% ammonium sulfate were done as described by Krause et al. (13). Further purification of the encoded gene product was carried out by anionexchange fast-performance liquid-chromatography on a MonoQ HR5/5 (Pharmacia, Inc.) column...

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Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium

Cited by 216 publications

References 15 publications

Non-Heme Fe(IV)–Oxo Intermediates

Non-Heme Fe(IV)–Oxo Intermediates

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S

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