THE GLUTAMINE LIGAND IN THE FERROUS IRON ACTIVE-SITE OF ISOPENICILLIN-N-SYNTHASE OF STREPTOMYCES-JUMONJINENSIS IS NOT ESSENTIAL FOR CATALYSIS

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron dependent d ioxygenase that catalyses the ring closure of delta-(L-alpha-aminoadip oyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. We previously used site-directed mutagenesis to identify in the IPNS of Streptomyces jum onjinensis two histidines and one aspartic acid that are essential for activity. The recent crystal structure of the IPNS of Aspergillus nid ulans establishes that these amino acids are iron ligands and reveals that the fourth ligand is the penultimate glutamine. The two histidine s and one aspartic acid are conserved in several classes of non-heme f errous iron dioxygenases, whereas the glutamine is present only in IPN Ss. In this paper we show that the penultimate glutamine in S. jumonji nensis IPNS Gln-328 is not essential for catalysis. In contrast, Gln-2 30 which is highly conserved among the above dioxygenases and is proxi mal to the active site is crucial for activity. (C) 1997 Federation of European Biochemical Societies.