Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of δ -(L-α-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37°C. Soluble expression was restored at a reduced temperature of 25°C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.Key words: isopenicillin N synthase, site-directed mutagenesis, glycine, Cephalosporium acremonium.

Replacement of Tyrosine-197 and the Corresponding Tyrosine-195 to Isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus Isopenicillin N Synthase

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.