Isopenicillin N epimerase

Abbreviations ACVS, 5-(L-a-aminoadipyl)-L-cysteinyl-D-valine Synthetase IPNS, isopenicillin N synthase IPNE, isopenicillin N epimerase DAOCS/ DACS, deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthase DAC-AT, acetyl CoA deacetylcephalosporin C O-acety 1 transferase DTT, dithiothreitol. 

C Lubbe, S Wolfe, AL Demain. Isopenicillin N epimerase activity in a high cephalo-... 

SE Jensen, DWS Westlake, S Wolfe. Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus. Can J Microbiol 29 1526-1531, 1983. 

S Usui, CA Yu. Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus. Biochim Biophys Acta 999 78-85, 1989. 

S Kovacevic, MB Tobin, JR Miller. The (3-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J Bacteriol 172 3952-3958, 1990. 

The biosynthesis of CPC is well known. The first step of CPC synthesis is the formation of the tripeptide S-(L-a-aminoadipyl)-L-cysteinlyl-D-valine (LLD-ACV) from L-a-aminoadipinic acid (AAA), L-cystein (CYS ) and D-valine (VAL) by the LLD-ACV synthetase (ACVS). LLD-ACV is converted with the enzyme isopenicillin AT-synthetase (cyclase) to isopenicillin N (IPN) and then with isopenicillin N-epimerase to penicillin N (PEN). The 6-ring is formed by deacetoxycephalosporin C-synthase (expandase). Deacetoxycephalosporin C (DAOC) is converted with deacetoxycephalosporin C-hydroxylase to decacetylcephalosporin C (DAC) and the latter with deacetylcephalosporin C-acyltransferase to CPC (Fig. 1). Except the amino acid and ACV, all others were monitored by on-line HPLC. 

Isopenicillin N is a precursor of penicillin, and synthesized from 5-(L-aminoadipoyl)-L-cysteinyl-D-valine by isopenicillin N synthetase11781. Isopenicillin N is then converted into penicillin N by isopenicillin N epimerase. Penicillin N is ring-expanded to deacetoxycepharosporin C by penicillin N expandase. The latter compound is... 

Isopenicillin N epimerase activity, demonstrated in the extract of Cepharosporium acremonium protoplasts was found to be very unstable(182). Usui and Yu,1S31, however, succeeded in purifying the enzyme to homogeneity after development of a simple assay procedure of the enzyme. They studied its enzymological properties [1831. The enzyme has a monomeric structure with a molecular mass of 47 000. The enzyme contains 1 mol of PLP per mol of protein. The enzyme shows a Vmax value of 3.93 pmol min-1 per mg and a Km of 0.30 him for isopenicillin N, whereas it shows a Vmax of 9.47 pmol min 1 per mg and a Km of 0.78 dim for penicillin N. The Keq value for the conversion between isopenicillin N and penicillin N is 1.09, which is in good agreement with the theoretical value. In addition to isopenicillin N and penicillin N, deacetoxycepharosporin C was epimerized only slowly the rate relative... 

Martin JF, Ullan RV, Casqueiro J Novel Genes Involved in Cephalosporin Biosynthesis The Three-component Isopenicillin N Epimerase System. In Brakhage AA, editor. Advances in Biochemical Engineering/Biotechnology. Berlin/Heidelberg Springer 2004, vol. 88, pp. 91-109. 

Lai L, Liras P. Castto JM, Martin JF Purification and characterization of the isopenicillin N epimerase from Nocordia lactamdHrons. J Gen Microbiol 1990 136 663-671. 

The metabolic fate of isopenicillin N differs depending on the organism. In C. acremonium Jayatilake et al. (269) demonstrated that lysed protoplasts could epimerise isopenicillin N to penicillin N, using a differential bioassay to detect the transformation. The epimerase was soluble, labile and intracellular. Intact protoplasts did not convert isopenicillin N to penicillin N. When penicillin N was added to lysed protoplasts generation of isopenicillin N could not be detected. Jensen et al. (265, 266) detected isopenicillin N epimerase activity in cell-free preparations of S. clavuligerus as discussed above. 

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