N‐terminal amino acid sequence and some properties of isopenicillin‐N synthetase from <i>Cephalosporium acremonium</i>

Baldwin, Jack E.; Gagnon, Jean; Ting, Hong-Hoi

doi:10.1016/0014-5793(85)80382-3

Cited by 30 publications

(8 citation statements)

References 7 publications

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“…It appears to be a monomer polypeptide since the SDS-denatured form of the enzyme showed the same relative molecular weight as the nondenatured form. Similar conclusions have been obtained with the IPN synthase of P. chrysogenum (24) and A. chrysogenum (2). The molecular weight of the acyltransferase (30,000) was slightly lower than that of the IPN synthase of P. chrysogenum (Mr, 39,000) (24) and A. chrysogenum (Mr, 38,416) (28).…”

Section: Discussionsupporting

confidence: 79%

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum

Álvarez

Cantoral

Barredo

et al. 1987

Antimicrob Agents Chemother

108

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The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-pQlyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 ± 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25°C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloroipercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.The 3-lactam-thiazolidine nucleus of penicillins is formed by cyclization of the tripeptide 8-(L-a-aminoadipyl)-Lcysteinyl-D-valine to form isopenicillin N (IPN), an intermediate in the biosynthetic pathway that has an L-aaminoadipyl side chain (Fig.

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Section: Discussionsupporting

confidence: 79%

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum

Álvarez

Cantoral

Barredo

et al. 1987

Antimicrob Agents Chemother

108

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“…It was shown that P. chrysogenum IPNS is strongly inhibited by glutathione and is also sensitive to cobalt inhibition (267). IPNS was purified to homogeneity from A. chrysogenum (27,142,251) and has subsequently been obtained from P. chrysogenum (267), A. nidulans (339), several actinomycetes such as S. clavuligerus (153), and the gram-negative bacterium Flavobacterium sp. (250).…”

Section: Structural Genes and Deduced Proteinsmentioning

confidence: 99%

“…(250). It was shown that two interconvertable forms of the enzyme exist, an oxidized state with a disulfide linkage and a reduced state (27).…”

Section: Structural Genes and Deduced Proteinsmentioning

confidence: 99%

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

Brakhage¹

1998

Microbiol Mol Biol Rev

211

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SUMMARY The most commonly used β-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as an end product by some fungi, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesized by both bacteria and fungi, e.g., by the fungus Acremonium chrysogenum (Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. Penicillin biosynthesis is catalyzed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC), and aatA (penDE). The genes are organized into a cluster. In A. chrysogenum, in addition to acvA and ipnA, a second cluster contains the genes encoding enzymes that catalyze the reactions of the later steps of the cephalosporin pathway (cefEF and cefG). Within the last few years, several studies have indicated that the fungal β-lactam biosynthesis genes are controlled by a complex regulatory network, e.g., by the ambient pH, carbon source, and amino acids. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of β-lactams and their physiological meaning for the producing fungi, and they can be expected to have a major impact on rational strain improvement programs. The knowledge of biosynthesis genes has already been used to produce new compounds.

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“…Although the reactions catalysing the biosynthesis of penicillins and cephalosporins have been demonstrated in cell-free systems, it is only very recently that we have begun to understand the mechanisms of some of these reactions. The enzyme catalysing the cyclization of the tripeptide L-a-aminoadipoyl-L-cysteinyl-D-valine to form isopenicillin N (isopenicillin N synthetase) has been purified to homogeneity from Cephalosporium acremonium (Acremonium chrysogenum) (Pang et al, 1984;Hollander et al, 1984;Baldwin et al, 1985) and this enzyme cloned into Escherichia coli (Samson et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

Baldwin

Adlington

Coates

et al. 1987

Biochemical Journal

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Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

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N‐terminal amino acid sequence and some properties of isopenicillin‐N synthetase from Cephalosporium acremonium

Cited by 30 publications

References 7 publications

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities

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