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Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

ABSTRACT: 1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

SUBMITTER: O'Sullivan J

PROVIDER: S-EPMC1161777 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium.

Project description:Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.

| S-EPMC1161778 | biostudies-other

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.

Project description:delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.

| S-EPMC1218779 | biostudies-other

Biochemical studies on the activity of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

Project description:The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.

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Structural studies on the reaction of isopenicillin N synthase with the substrate analogue delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alpha-aminobutyrate.

Project description:Isopenicillin N synthase (IPNS) is a non-haem iron(II) oxidase which catalyses the biosynthesis of isopenicillin N from the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). Herein we report crystallographic studies to investigate the reaction of IPNS with the truncated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate (ACAb). It has been reported previously that this analogue gives rise to three beta-lactam products when incubated with IPNS: two methyl penams and a cepham. Crystal structures of the IPNS-Fe(II)-ACAb and IPNS-Fe(II)-ACAb-NO complexes have now been solved and are reported herein. These structures and modelling studies based on them shed light on the diminished product selectivity shown by IPNS in its reaction with ACAb and further rationalize the presence of certain key residues at the IPNS active site.

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Substrate specificity of L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase from Cephalosporium acremonium: demonstration of the structure of several unnatural tripeptide products.

Project description:Potential substrates for L-delta-(alpha-aminoadipoyl)-L-(cysteinyl)-D-valine (ACV) synthetase were initially identified using both the amino-acid-dependent ATP<-->pyrophosphate exchange reaction catalysed by the enzyme and the incorporation of 14C-radiolabelled cysteine and valine into potential peptide products. S-Carboxymethylcysteine was an effective substitute for alpha-aminoadipate and both allylglycine and vinylglycine could substitute for cysteine, indicating that the thiol group of cysteine is not essential for peptide formation. L-allo-Isoleucine but not L-isoleucine substituted effectively for valine. The structures of the presumed peptide products derived from these amino acids were confirmed by combined use of electrospray-ionization m.s. (e.s.m.s.) and 1H n.m.r. These results clearly indicate that, in common with other peptide synthetases, but in contrast with ribosomal peptide synthesis, ACV synthetase has a relatively broad substrate specificity.

| S-EPMC1137089 | biostudies-other

A study of the biosynthesis of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in a beta-lactam-negative mutant of Cephalosporium acremonium.

Project description:A cell-free extract of Cephalosporium acremonium (Takeda N-2) was obtained that synthesized the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and also the dipeptide delta-(L-alpha-aminoadipyl)-L-cysteine from the corresponding L-amino acids.

| S-EPMC1152170 | biostudies-other

Ethyl 2-diethyl-amino-4-oxo-3,5-diphenyl-4,5-dihydro-3H-pyrrolo-[3,2-d]pyrimidine-7-carboxyl-ate.

Project description:In the title compound, C(25)H(26)N(4)O(3), the two fused pyrrolo-[3,2-d]pyrimidine rings form a dihedral angle of 3.7?(2)°. The two substituent phenyl rings are twisted with respect to the pyrrole and pyrimidine rings, making dihedral angles of 57.2?(2) and 69.0?(2)°, respectively. The ethyl and eth-oxy groups are disordered over two positions; the site occupancies are 0.53?(1) and 0.47?(1) for ethyl, and 0.63?(1) and 0.37?(1) for eth-oxy. The crystal packing features C-H?O hydrogen bonds.

| S-EPMC3254465 | biostudies-literature

Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

Project description:1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.

| S-EPMC1161726 | biostudies-other

2,4-Diphenyl-4,5-dihydro-3H-pyrido[2,3-b][1,4]diazepine.

Project description:The asymmetric unit of the title compound, C(20)H(17)N(3), contains two crystallographically independent mol-ecules (A and B). In mol-ecule A, the two benzene rings form dihedral angles of 74.12 (7) and 7.83 (7)° with the pyridine ring, while in mol-ecule B these angles are 77.48 (7) and 21.50 (7)°. The seven-membered heterocyclic ring adopts a boat conformation in both mol-ecules. In the crystal structure, each of the independent mol-ecules forms a centrosymmetric R(2) (2)(8) dimer linked by paired N-H⋯N hydrogen bonds. The crystal structure is further stabilized by inter-molecular C-H⋯N hydrogen bonds and C-H⋯π inter-actions.

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Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.

Project description:Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.

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