Site-directed mutagenesis of proline-285 to leucine in Cephalosporium acremonium isopenicillin N synthase affects catalysis and increases soluble expression at higher temperatures

The conversion of delta-(L-alpha -aminoadipyl)-L-cysteinyl-D-valine (ACV) t o isopenicillin N is dependant on the catalytic action of isopenicillin N - synthase (IPNS), an important enzyme in the penicillin and cephalosporin b iosynthetic pathway. One of the amino acid residues suggested by the Asperg illus nidulans IPNS crystal structure for interaction with the valine isopr opyl group of ACV is proline-283. Site-directed mutagenesis of the correspo nding proline-285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher tempe ratures in a heterologous E. coli host.