Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which
catalyses the oxidative conversion of anddelta;-(L-andalpha;-aminoadipoyl)-L-cysteinyl-D-valine (ACV)
to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the
complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also
known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The
crystal structure of IPNS (Aspergillus nidulans) active site (in the absence of ACV)
revealed an octahedrally coordinated manganese atom surrounded by these four protein
ligands and two water molecules.
The role of the four conserved metal binding ligands was investigated using site
directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was
not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the
three remaining metal ligands was indispensable for catalytic activity. Additionally, it
was demonstrated that the conserved Asp216 residue may be substituted by a glutamate
residue (D216E) with significant retention of catalytic activity. Crystallographic and
spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a
similar way to wild-type IPNS.
The inactivation of wild-type IPNS was examined under in vitro assay conditions.
This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway via Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was
substantially reduced by the inclusion of catalase in the assay mixture, thus indicating
that oxidative IPNS inactivation results (in part) from the generation of hydrogen
peroxide in solution. Inactivation was also accompanied by a slow fragmentation of
intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate
polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the
fragmentation resulted from at least two cleavage sites within the active site (between
Asp216-Val217 and Val272-Lys273).</p