INVOLVEMENT OF A 3RD HISTIDINE IN THE FERROUS ACTIVE-SITE OF ISOPENICILLIN-N SYNTHASE OF CEPHALOSPORIUM-ACREMONIUM REPUDIATED BY RECOMBINANT DOUBLE HISTIDINE MUTANTS

Site-directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential h istidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking hi stidine at positions 216 and 272 clearly supports the essential role t hese two histidines play in iron binding. However, nuclear magnetic re sonance (NMR) studies have indicated that there could be three histidi ne residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS ge nes containing mutations at two histidine codons were created by in vi tro cloning of fragments from the expression vectors bearing the respe ctive cIPNS genes each with a single histidine mutation at positions H 49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre o f cIPNS, then one of the constructed double histidine mutants would ha ve lost its enzymatic activity. However, analysis of the cIPNS activit ies of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third hist idine can be repudiated.