Conversion of isopenicillin N into penicillin N in cell-free extracts of <i>Cephalosporium acremonium</i>

Jayatilake, Gamini S.; Huddleston, Joyce A.; Abraham, E. P.

doi:10.1042/bj1940645

Cited by 48 publications

(25 citation statements)

References 10 publications

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“…Since S. clavuligerus cell-free extracts are routinely centrifuged at high speed and stored at -20°C before assay, the cyclization and racemase activities must both be "soluble" and quite stable. Racemase activity in C. acremonium is difficult to demonstrate in cell-free extracts and is suggested to be very labile and possibly membrane-bound1, 5,7,8,22,23). SAWADA et al have proposed that cyclization activity in C. acremonium is also membrane-bound based on the stimulatory effects of Triton X-100 and sonication8'.…”

Section: Resultsmentioning

confidence: 99%

Cyclization of .DELTA.-(L-.ALPHA.-aminoadipyl)-L-cysteinyl-D-valine to penicillins by cell-free extracts of Streptomyces clavuligerus.

1982

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Cell-free extracts prepared by sonication of Streptomyces clavuligerus cyclized 6-(L-aaminoadipyl)-L-cysteinyl-D-valine (ACV) into a penicillin-type antibiotic. The antibacterial spectrum of this antibiotic suggested it was a mixture of isopenicillin N and penicillin N indicating that both cyclization and racemase activities were present. Cyclization activity was optimal in extracts prepared from 48 hours cultures. Extracts incubated at 20°C produced antibiotic for 2 hours before activity ceased. Cyclization activity showed an absolute requirement for dithiothreitol (DTT) and 02 and was stimulated by ascorbic acid and FeSO4. No requirement for ATP was observed.

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Section: Resultsmentioning

confidence: 99%

Cyclization of .DELTA.-(L-.ALPHA.-aminoadipyl)-L-cysteinyl-D-valine to penicillins by cell-free extracts of Streptomyces clavuligerus.

1982

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“…Typically, onset of cephamycin C biosynthesis in control cultures coincides with the end of growth phase when carbon or nitrogen sources become limiting (18,19). Production of epimerase and expandase in "- 10 VOL. 172,1990 on May 11, 2018 by guest http://jb.asm.org/ Downloaded from S. lactamdurans, for example, may be further repressed by growing the culture in medium supplemented with glucose or a nitrogen source (ammonia) (4).…”

Section: Resultsmentioning

confidence: 99%

“…1) (7,11). The first step that commits the pathway to the production of cephalosporinlike antibiotics is the isomerization of isopenicillin N, the product of isopenicillin N synthetase (cyclase), to penicillin N (10,12). This activity has also been referred to as epimerase and is not found in organisms (e.g., Penicillium chrysogenum) that produce hydrophobic penicillins (7).…”

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confidence: 99%

The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus

1990

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Isopenicihlin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined. By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the P-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment. The epimerase gene (cel)) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously. celD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity. Western immunoblots demonstrated that a protein comigrated with purified S. clavuligerus epimerase at 44 kilodaltons. cel) and cejE were separated by an 81-base-pair segment. The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region. Primer extension analysis of S. clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease Si protection assays showed that a single message may code for epimerase, expandase, and another ulnknown protein. When cef) and cejE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E. coli.,-Lactam antibiotics are distinguished by the ,3-lactam ring plus either a five-membered thiazolidine ring (present in the penicillins) or the "expanded" six-membered dihydrothiazine structure (found in the cephalosporins). This is the main chemical difference between the core structures of these antibiotics. The pathway for these two classes of 13-lactams have common steps in their synthesis up to the production of isopenicillin N ( Fig. 1) (7, 11). The first step that commits the pathway to the production of cephalosporinlike antibiotics is the isomerization of isopenicillin N, the product of isopenicillin N synthetase (cyclase), to penicillin N (10, 12). This activity has also been referred to as epimerase and is not found in organisms (e.g., Penicillium chrysogenum) that produce hydrophobic penicillins (7).In a previous report (13) we characterized the gene (cefE) for deacetoxycephalosporin C synthetase (DAOCS; expandase) and expressed the gene in Escherichia coli. These E. coli extracts contained expandase activity. The gene was isolated from a cosmid library with a single hybridization probe based on the N-terminal amino acid sequence of the purified protein and the strict codon bias observed for streptomycetes (24). We have also shown by differential hybridization to these cosmids that genes for the cyclase and expandase enzymes are closely linked in S. clavuligerus (13,21). This potential for linkage of all the genes for the biosynthesis of cephalosporins led us to characterize the molecular organization of the epimerase ...

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“…The enzyme was so labile that it could not be characterized further (Liibbe et al, 1986). The extreme lability of the epimerase of C. acrernonium has been confimed by other groups (Baldwin et al, 1981 ;Jayatilake et al, 1981) who found activity only in freshly prepared cell-free extracts. The epimerase of S. claudigerus appears to be more stable and could be partially purified, although the protein band corresponding to the theoretical Mr was barely detectable in polyacrylamide gels (Jensen et af., 1983).…”

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confidence: 91%

Purification and characterization of the isopenicillin N epimerase from Nocardia lactamdurans

Láiz

Liras

Castro

et al. 1990

Journal of General Microbiology

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The biosynthesis of cephalosporirns by Cephalosporium acremonium (syn. Acremonium chrysogenum) and cephamycins by Nocardia lactamdurans (syn. Streptomyces lactamdurans) and Streptomyces clavuligerus takes place through similar biosynthetic pathways (reviewed by Martin & Liras, 1985). In both cases, the first step involves the formation of the tripeptide &(L-a-aminoadipy1)-L-cysteinyl-D-valine (ACV) by the ACV synthetase complex, followed by cyclization of ACV to isopenicillin N (IBN). Conversion of IPN to deacetoxycephalosporin C (the first intermediate carrying the six-membered dihydrothiazine ring characteristic of the cephalosporin molecules) is catalysed by two enzymes : IPN epimerase, which epimerizes IPN to penicillin N and deacetoxycephalosporin C synthase (expandase) which expands the five-membered thiazolidine ring into the six-membered dihydrothiazine ring. Deacetoxycephalosporin C is later converted into either cephalosporin C or cephamycins by a series of enzymic reactions (Martin et al., 1986; Martin & Liras, 1989).IPN epimerase, which converts IPN into penicillin N by changing the L-a-aminoadipic acid side chain to the DAbbreviations : ACV, S-(L-a-aminoadipyl>L-cysteinyl-Dvaline ; IPN, isopenkillin N ; PP, pyridoxal phosphate. configuration ( Fig. 1) was first detected in C. acremonim (Konomi et al., 1979;Sawada et al., 1980). The enzyme was so labile that it could not be characterized further (Liibbe et al., 1986). The extreme lability of the epimerase of C. acrernonium has been confimed by other groups (Baldwin et al., 1981 ; Jayatilake et al., 1981) who found activity only in freshly prepared cell-free extracts. The epimerase of S. claudigerus appears to be more stable and could be partially purified, although the protein band corresponding to the theoretical Mr was barely detectable in polyacrylamide gels (Jensen et af., 1983). It seems, therefore, that IPN epimerases ~E-QIII other cephamycin-producing micro-organisms might possess properties (including higher stability) that may allow a complete characterization of this enzyme.Little is known about the molecular mechanism of amino acid epimerization during antibiotic biosynthesis. Some of the best-known epimerases involved in peptide antibiotic biosynthesis require ATP as an activating cofactor (Takahashi et al., 1971).

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Conversion of isopenicillin N into penicillin N in cell-free extracts of Cephalosporium acremonium

Abstract: In a cell-free system prepared by osmotic lysis of protoplasts of Cephalosporium acremonium, isopenicillin N is converted into penicillin N. The epimerase activity of the system is labile.

Cited by 48 publications

References 10 publications

Cyclization of .DELTA.-(L-.ALPHA.-aminoadipyl)-L-cysteinyl-D-valine to penicillins by cell-free extracts of Streptomyces clavuligerus.

Cyclization of .DELTA.-(L-.ALPHA.-aminoadipyl)-L-cysteinyl-D-valine to penicillins by cell-free extracts of Streptomyces clavuligerus.

The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus

Purification and characterization of the isopenicillin N epimerase from Nocardia lactamdurans

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