Isopenicillin N Synthetase of
            <i>Penicillium chrysogenum</i>
            , an Enzyme That Converts δ-(
            <scp>l</scp>
            -α-Aminoadipyl)-
            <scp>l</scp>
            -Cysteinyl-
            <scp>d</scp>
            -Valine to Isopenicillin N

Ramos, Filomena R.; López-Nieto, Manuel J.; Martı́n, Juan F.

doi:10.1128/aac.28.3.464-a

Cited by 21 publications

(36 citation statements)

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“…Isopenicillin N synthase (cyclase) was determined in cell extracts of P. chrysogenum as described by Ramos et al (21) after filtration through a Sephadex G-25 column to remove residual penicillin G and other low-molecular-weight molecules in the extract.…”

Section: Analysis Of Intracellular Concentration Of Amino Acidsmentioning

confidence: 99%

“…The recent development of reliable assay methods to quantify the tripeptide ACV (11) and to measure the activities of isopenicillin N synthase (21) and penicillin acyltransferase (E. Alvarez and J. F. Martin, unpublished data) prompted us to study the effect of glucose on the synthesis * Corresponding author. and activity of the different enzymes involved in penicillin biosynthesis, as well as on the availability of precursor amino acids for tripeptide biosynthesis.…”

mentioning

confidence: 99%

“…1). ACV is then cyclized to form isopenicillin N by the action of isopenicillin N synthase, an enzyme that has been purified to near homogeneity from extracts of P. chrysogenum (19,21). In the last step of penicillin biosynthesis, the a-aminoadipyl side chain of isopenicillin N is exchanged for phenylacetic acid, which is previously activated in the form of phenylacetyl coenzyme A (CoA) (20; B. Spencer and C. Maung, Biochem.…”

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confidence: 99%

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Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Revilla¹,

Ramos²,

López-Nieto³

et al. 1986

J Bacteriol

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The content of a-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into a-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of a-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing at-aminoadipic acid accumulation. The repression of a-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of oa-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts a-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.Glucose exerts a negative control on the biosynthesis of penicillin in Penicillium chrysogenum (17,22) similar in many aspects to carbon catabolite regulation of the biosynthesis of cephalosporins in Acremonium chrysogenum (3,24) and cephamycins in Streptomyces clavuligerus (1,23) and Streptomyces lactamdurans (6). Glucose (28 to 140 mM) produces a concentration-dependent repression of the incorporation of ['4C]valine into penicillin but does not have an inhibitory effect on such incorporation by enzymes formed before glucose addition (22). The total activity of the penicillin-synthesizing enzymes in P. chrysogenum is repressed by glucose (22). Derepression of penicillin biosynthesis occurs after depletion of glucose. However, the overall biosynthetic activity of the pathway in vivo is determined by the availability of precursors of the L-a-aminoadipyl-Lcysteinyl-D-valine (ACV) tripeptide and by the activities of the (at least) three enzymes involved in penicillin biosynthesis.The first well-established intermediate in the penicillin biosynthetic pathway, ACV (2, 11), appears to be formed by a sequential condensation of the three precursor amino acids (8) (Fig. 1). ACV is then cyclized to form isopenicillin N by the action of isopenicillin N synthase, an enzyme that has been purified to near homogeneity from extracts of P. chrysogenum (19,21). In the last step of penicillin biosynthesis, the a-aminoadipyl side chain of isopenicillin N is exchanged for phenylacetic acid, which is previously activated in the form of phenylacetyl coenzyme A (CoA) (20; B. Spencer and C. Maung, Biochem. J. 118:29p-30p, 1970).The recent development of reliable assay methods to quantify the tripeptide ACV (11) and to measure the activities of isopenicillin N synthase (21) and penicillin acyltransferase (E.

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Section: Analysis Of Intracellular Concentration Of Amino Acidsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Revilla¹,

Ramos²,

López-Nieto³

et al. 1986

J Bacteriol

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“…IPNS synthesis is therefore not associated with the logarithmic growth rate and is subject to strict regulatory control. Formation of IPNS (36) and onset of penicillin production are also known to occur after the rapid growth phase in a highly productive strain of P. chrysogenum (33), but a high level of IPNS activity was still present thereafter throughout fermentation. In contrast, rapid disappearance of IPNS activity was shown to occur in C. acremonium (36) and S. clavuligerus (22).…”

Section: Discussionmentioning

confidence: 99%

“…This enzyme has been well characterized in the filamentous fungi Cephalosporium acremonium (19,31) and Penicillium chrysogenum (33) and in the actinomycetes Streptomyces clavuligerus (21) and S. lactamdurans (13) and is readily available, since the IPNS genes from these different microorganisms have been cloned, se- (volume of air to fermentor volume per minute). 02 consumption, redox potential, and pH were monitored throughout fermentation.…”

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confidence: 99%

Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154

et al. 1989

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The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level 13-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including a-aminoadipic acid and cysteine. The Km for a-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a P-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of t3-lactams in bacteria.

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Recombinant microorganisms for industrial production of antibiotics

Dı́ez

Mellado

Rodríguez

et al. 1997

Biotechnol. Bioeng.

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The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7‐aminocephalosporanic acid (7‐ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl‐7‐aminodeacetoxycephalosporanic acid (adipyl‐7‐ADCA) and adipyl‐7‐ACA, compounds that can be transformed into the economically relevant 7‐ADCA and 7‐ACA intermediates. Escherichia coli expression of the genes encoding D‐amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7‐ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 216–226, 1997.

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Isopenicillin N Synthetase of Penicillium chrysogenum , an Enzyme That Converts δ-( l -α-Aminoadipyl)- l -Cysteinyl- d -Valine to Isopenicillin N

Cited by 21 publications

References 0 publications

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum

Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154

Recombinant microorganisms for industrial production of antibiotics

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