PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
 
J Bacteriol. 1990 October; 172(10): 5908–5914.
PMCID: PMC526911

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

Abstract

The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alonso MJ, Bermejo F, Reglero A, Fernández-Cañn JM, González de Buitrago G, Luengo JM. Enzymatic synthesis of penicillins. J Antibiot (Tokyo) 1988 Aug;41(8):1074–1084. [PubMed]
  • Alvarez E, Cantoral JM, Barredo JL, Díez B, Martín JF. Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum. Antimicrob Agents Chemother. 1987 Nov;31(11):1675–1682. [PMC free article] [PubMed]
  • Barredo JL, van Solingen P, Díez B, Alvarez E, Cantoral JM, Kattevilder A, Smaal EB, Groenen MA, Veenstra AE, Martín JF. Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-acid-acyltransferase gene of Penicillium chrysogenum. Gene. 1989 Nov 30;83(2):291–300. [PubMed]
  • Carr LG, Skatrud PL, Scheetz ME, 2nd, Queener SW, Ingolia TD. Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene. 1986;48(2-3):257–266. [PubMed]
  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Díez B, Barredo JL, Alvarez E, Cantoral JM, van Solingen P, Groenen MA, Veenstra AE, Martín JF. Two genes involved in penicillin biosynthesis are linked in a 5.1 kb SalI fragment in the genome of Penicillium chrysogenum. Mol Gen Genet. 1989 Sep;218(3):572–576. [PubMed]
  • Gatenbeck S. Acyl CoA:6-aminopenicillanic acid acyltransferase. Methods Enzymol. 1975;43:474–476. [PubMed]
  • Gatenbeck S, Brunsberg U. Biosynthesis of penicillins. I. Isolation of a 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum. Acta Chem Scand. 1968;22(3):1059–1061. [PubMed]
  • Ingolia TD, Queener SW. Beta-lactam biosynthetic genes. Med Res Rev. 1989 Apr-Jun;9(2):245–264. [PubMed]
  • Kovacevic S, Weigel BJ, Tobin MB, Ingolia TD, Miller JR. Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J Bacteriol. 1989 Feb;171(2):754–760. [PMC free article] [PubMed]
  • Luengo JM, Iriso JL, López-Nieto MJ. Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay. J Antibiot (Tokyo) 1986 Nov;39(11):1565–1573. [PubMed]
  • Luengo JM, Iriso JL, López-Nieto MJ. Direct enzymatic synthesis of natural penicillins using phenylacetyl-CoA: 6-APA phenylacetyl transferase of Penicillium chrysogenum: minimal and maximal side chain length requirements. J Antibiot (Tokyo) 1986 Dec;39(12):1754–1759. [PubMed]
  • Montenegro E, Barredo JL, Gutiérrez S, Díez B, Alvarez E, Martín JF. Cloning, characterization of the acyl-CoA:6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene. Mol Gen Genet. 1990 May;221(3):322–330. [PubMed]
  • Rambosek J, Leach J. Recombinant DNA in filamentous fungi: progress and prospects. Crit Rev Biotechnol. 1987;6(4):357–393. [PubMed]
  • Schoner BE, Belagaje RM, Schoner RG. Translation of a synthetic two-cistron mRNA in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8506–8510. [PubMed]
  • Smith DJ, Burnham MK, Bull JH, Hodgson JE, Ward JM, Browne P, Brown J, Barton B, Earl AJ, Turner G. Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J. 1990 Mar;9(3):741–747. [PubMed]
  • Weigel BJ, Burgett SG, Chen VJ, Skatrud PL, Frolik CA, Queener SW, Ingolia TD. Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans. J Bacteriol. 1988 Sep;170(9):3817–3826. [PMC free article] [PubMed]
  • Whiteman PA, Abraham EP, Baldwin JE, Fleming MD, Schofield CJ, Sutherland JD, Willis AC. Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans. FEBS Lett. 1990 Mar 26;262(2):342–344. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)