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.
The tripeptide 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicilium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2' and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25C and 7.8, respectively. The reaction required 02 and was stimulated by increasing the dissolved oxygen concentration of the reaction mixture. Purification of the enzyme to a single major band in polyacrylamide gel electrophoresis was achieved by protamine sulfate precipitation, ammonium sulfate fractionation (50 to 80% of saturation), DEAE-Sephacel chromatography, and gel filtration on Sephacryl S-200. The estimated molecular weight was 39,000 + 1,000.The apparent Km of isopenicillin N synthetase for 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine was 0.13 mM. The enzyme activity was strongly inhibited by glutathione, which acts as a competitive inhibitor. A good correlation was observed between the isopenicillin N synthetase activity in extracts of four different strains of P. chrysogenum (with widely different penicillin-producing capability) and the amount of penicillin production by these strains.The enzymes carrying out the conversion of the precursor amino acids (a-aminoadipic acid, cysteine, and valine) into penicillin G have not been purified and characterized in spite of the industrial importance of this antibiotic (6).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.