Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity.

Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe(2+)-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli. When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.