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. 1997 Oct 1;327(Pt 1):185–191. doi: 10.1042/bj3270185

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

H B Theilgaard 1, K N Kristiansen 1, C M Henriksen 1, J Nielsen 1
PMCID: PMC1218779  PMID: 9355751

Abstract

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.

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Selected References

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  1. Baldwin J. E., Bird J. W., Field R. A., O'Callaghan N. M., Schofield C. J., Willis A. C. Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. Evidence for the presence of phosphopantothenate in ACV synthetase. J Antibiot (Tokyo) 1991 Feb;44(2):241–248. doi: 10.7164/antibiotics.44.241. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Christensen L. H., Henriksen C. M., Nielsen J., Villadsen J., Egel-Mitani M. Continuous cultivation of Penicillium chrysogenum. Growth on glucose and penicillin production. J Biotechnol. 1995 Sep 29;42(2):95–107. doi: 10.1016/0168-1656(95)00056-v. [DOI] [PubMed] [Google Scholar]
  4. Cohen G., Argaman A., Schreiber R., Mislovati M., Aharonowitz Y. The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis. J Bacteriol. 1994 Feb;176(4):973–984. doi: 10.1128/jb.176.4.973-984.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Díez B., Gutiérrez S., Barredo J. L., van Solingen P., van der Voort L. H., Martín J. F. The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes. J Biol Chem. 1990 Sep 25;265(27):16358–16365. [PubMed] [Google Scholar]
  6. Jensen S. E., Wong A., Rollins M. J., Westlake D. W. Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. J Bacteriol. 1990 Dec;172(12):7269–7271. doi: 10.1128/jb.172.12.7269-7271.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kadima T. A., Jensen S. E., Pickard M. A. Production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine by entrapped ACV-synthetase from Streptomyces clavuligerus. J Ind Microbiol. 1995 Jan;14(1):35–40. doi: 10.1007/BF01570064. [DOI] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. MacCabe A. P., van Liempt H., Palissa H., Unkles S. E., Riach M. B., Pfeifer E., von Döhren H., Kinghorn J. R. Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway. J Biol Chem. 1991 Jul 5;266(19):12646–12654. [PubMed] [Google Scholar]
  10. Nielsen J., Jørgensen H. S. Metabolic control analysis of the penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum. Biotechnol Prog. 1995 May-Jun;11(3):299–305. doi: 10.1021/bp00033a010. [DOI] [PubMed] [Google Scholar]
  11. Nielsen J. Metabolic control analysis of biochemical pathways based on a thermokinetic description of reaction rates. Biochem J. 1997 Jan 1;321(Pt 1):133–138. doi: 10.1042/bj3210133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Perry D., Abraham E. P., Baldwin J. E. Factors affecting the isopenicillin N synthetase reaction. Biochem J. 1988 Oct 1;255(1):345–351. [PMC free article] [PubMed] [Google Scholar]
  13. Schwecke T., Aharonowitz Y., Palissa H., von Döhren H., Kleinkauf H., van Liempt H. Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. Eur J Biochem. 1992 Apr 15;205(2):687–694. doi: 10.1111/j.1432-1033.1992.tb16830.x. [DOI] [PubMed] [Google Scholar]
  14. Smith D. J., Earl A. J., Turner G. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J. 1990 Sep;9(9):2743–2750. doi: 10.1002/j.1460-2075.1990.tb07461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zhang J. Y., Demain A. L. Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins. Biochem Biophys Res Commun. 1990 Jun 29;169(3):1145–1152. doi: 10.1016/0006-291x(90)92015-r. [DOI] [PubMed] [Google Scholar]
  16. Zhang J., Demain A. L. Regulation of ACV synthetase in penicillin- and cephalosporin-producing microorganisms. Biotechnol Adv. 1991;9(4):623–641. doi: 10.1016/0734-9750(91)90735-e. [DOI] [PubMed] [Google Scholar]
  17. Zhang J., Wolfe S., Demain A. L. Biochemical studies on the activity of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. Biochem J. 1992 May 1;283(Pt 3):691–698. doi: 10.1042/bj2830691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. van Liempt H., von Döhren H., Kleinkauf H. delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase. J Biol Chem. 1989 Mar 5;264(7):3680–3684. [PubMed] [Google Scholar]

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