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 delta-(L-alpha -aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in t he 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 investigate d by site-directed mutagenesis to study the effect of the substitution on c atalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its solub le expression in a heterologous host at 37 degreesC. Soluble expression was restored at a reduced temperature of 25 degreesC, and thus, it is possible that these glycine residues may have a role in maintaining the local prote in structure and are critical for the soluble expression of cIPNS.