Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium

Nature ◽  
1985 ◽  
Vol 318 (6042) ◽  
pp. 191-194 ◽  
Author(s):  
S. M. Samson ◽  
R. Belagaje ◽  
D. T. Blankenship ◽  
J. L. Chapman ◽  
D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cephalosporium Acremonium ◽  
Sequence Determination ◽  
Expression In Escherichia Coli ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Studies on the ring-cyclization and ring-expansion enzymes of β-lactam biosynthesis in Cephalosporium acremonium

1983 ◽  
Vol 29 (5) ◽  
pp. 488-496 ◽  
Author(s):  
Jutta Kupka ◽  
Yong-Qiang Shen ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Ring Expansion ◽  
Exchange Chromatography ◽  
Cephalosporium Acremonium ◽  
Competitive Inhibitors ◽  
Isopenicillin N Synthetase ◽  
Soluble Enzymes ◽  
Temperature Optima ◽  
Isopenicillin N

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine required reduction to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including α-ketoglutarate, were inactive. In addition to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-δ-(L-α-aminoadipyl) L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deactoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

Gene ◽  
1988 ◽  
Vol 62 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Brenda K. Leskiw ◽  
Yair Aharonowitz ◽  
Moshe Mevarech ◽  
Saul Wolfe ◽  
Leo C. Vining ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Streptomyces Clavuligerus ◽  
Sequence Determination ◽  
Isopenicillin N Synthetase ◽  
Nucleotide Sequence Determination ◽  
Isopenicillin N

scholarly journals Purification of isopenicillin N synthetase

1984 ◽  
Vol 222 (3) ◽  
pp. 789-795 ◽  
Author(s):  
C P Pang ◽  
B Chakravarti ◽  
R M Adlington ◽  
H H Ting ◽  
R L White ◽  
...  
Keyword(s):  
Streptomyces Clavuligerus ◽  
Protein Band ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Synthetase Activity ◽  
Cephalosporium Acremonium ◽  
Major Protein Band ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.

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