Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N

Abstract

Cell-free cyclization of S-(L-a-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N by lytic enzyme extracts of Cephalosporium acremonium M-0198 was stimulated by ferrous ions. The optimum concentration of FeSO4 was 80 ,uM. No additional stimulation was observed with ascorbate, adenosine 5'-triphosphate, or a-ketoglutarate, but Triton X-100 and sonication of the extracts increased activity. ZnSO4 was very inhibitory to enzyme activity; CUSO4 was somewhat less inhibitory, and the least effective of the three was MnCl2. The dimer of the tripeptide was converted to a penicillin that has the biological spectrum of isopenicillin N, and this reaction was also stimulated by FeSO4. We found that sonication can be used directly to prepare extracts with cyclization activity from mycelia, without preparing protoplast lysates. The kinetics of cyclase appearance and disappearance during fermentation were similar to those of ring-expansion activity, i.e., enzyme appeared and peaked 13 h after growth ceased and then disappeared.Both our group (7) and Abraham's group (9) have shown, independently, that 6-(L-a(aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) is cyclized to isopenicillin N by extracts of Cephalosporium acremonium (Fig. 1). O'Sullivan et al. (9) isolated the product of the cell-free conversion and determined that the side chain of the penicillin was L-a-aminoadipic acid. Because oxidation of deacetoxycephalosporin C to deacetylcephalosporin C is stimulated by ferrous ions, ascorbate, and a-ketoglutarate (12) and because oxidative ring-expansion of penicillin N to deacetoxycephalosporin C is stimulated by ferrous ions and ascorbate (4, 10) we checked the effect of these additives on the oxidative ring-closure reaction. We also tested the effect of Triton X-100, which, we found, stimulates ring-expansion by these same cell-free extracts (11).We also wanted to determine whether the dimer of LLD-tripeptide, i.e., bis[S-(L-a-aminoadipyl)-L-cysteinyl-D-valine]disulfide, could be converted to isopenicillin N. This dimer is known to be produced by certain blocked mutants of C. acremonium (5)

Penicillin formation by cell-free extracts of Streptomycesclavuligerus. Behaviour of aminoadipyl-modified analogs of the natural peptide precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV)

“…In view of these various observations, analogs of ACV were synthesized in which the 6-(L-a-aminoadipyl) moiety was replaced by: p-(L-aspartyl) (6), y-(L-glutamyl) (7), 6-(D-aaminoadipyl) (S), adipyl (9), N-acetyl-6-(L-a-aminoadipyl) (lo), and glycyl-6-(L-a-aminoadipyl) (11). Each of these peptides was then subjected to the action of the cyclizing enzyme of S. clavuligerus.…”

Beta-lactam biosynthesis in a gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154

“…The detection of IPNS associated with the membrane in Western blot analysis was striking, since all forms of IPNS studied have been found in the soluble fraction upon rupture of the cells. Nevertheless, some researchers have reported latent membrane-bound IPNS activity in cell-free extracts containing the membrane fraction in S. clavuligerus (22) and C. acremonium (36,37). The membrane-associated Flavobacterium enzyme was not active when tested in Triton X-100-solubilized extracts, although the detergent concentration used for solubilization (0.02%) did not inhibit the activity of the purified enzyme.…”

“…IPNS synthesis is therefore not associated with the logarithmic growth rate and is subject to strict regulatory control. Formation of IPNS (36) and onset of penicillin production are also known to occur after the rapid growth phase in a highly productive strain of P. chrysogenum (33), but a high level of IPNS activity was still present thereafter throughout fermentation. In contrast, rapid disappearance of IPNS activity was shown to occur in C. acremonium (36) and S. clavuligerus (22).…”

Purification and characterization of the isopenicillin N epimerase from Nocardia lactamdurans

“…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).…”