Functional Analysis of Conserved Histidine Residues in Cephalosporium acremonium Isopenicillin N Synthase by Site-directed Mutagenesis

Abstract

The isopenicillin N synthase of Cephalosporium acremonium (cIPNS) involves a catalytically important non-heme iron which is coordinated credibly to histidine residues. A comparison of the IPNS genes from various microbial sources indicated that there are seven conserved histidine residues. These were individually replaced by leucine residues through site-directed mutagenesis, and the sites of mutation were confirmed by DNA sequencing. The seven mutant genes were cloned separately into the vector pET24d for expression in Escherichia coli BL21 (DE3), and the proteins were expressed as soluble enzymes. All the resulting mutant enzymes obtained have mobilities of approximately 38 kDa, identical with the wild-type enzyme on SDS-polyacrylamide gel electrophoresis, and were also reactive to cIPNS antibodies. The enzymes were purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography, and these were analyzed for enzyme activity. A group of mutant enzymes, H49L, H64L, H116L, H126L, and H137L, were found to be enzymatically active with reduced activities of 16-93.7%, indicating that they are not essential for catalysis. Two of the mutant enzymes, H216L and H272L, were found to have lost their enzymatic activity completely, indicating that both His-216 and His-272 are crucial for catalysis. It is suggested that these histidines are likely to serve as ligands for binding to the non-heme iron in the IPNS active site. Alignment of the amino acid sequence of IPNS to related non-heme Fe(2+)-requiring enzymes indicated that the two essential histidine residues correspond to two invariant residues located in highly homologous regions. The conservation of the two closely located histidine residues indicates the possible conservation of similar iron-binding sites in these enzymes.

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

“…A role for histidines as putative iron ligands has recently been defined on the basis of the crystal structure of isopenicillin N synthase from Aspergillus nidulans [ 151 and by site-directed mutagenesis of the isopenicillin N synthase enzymes from Streptomyces jurnonjinensis and Cephalosporium acremorzium [16,171. Using the three-dimensional structure of isopenicillin N synthase, it was concluded that the manganese ion was attached by His214, His270, Asp216, and Gln330.…”

The 2‐His‐1‐Carboxylate Facial Triad — An Emerging Structural Motif in Mononuclear Non‐Heme Iron(II) Enzymes

“…1) [ I , 21. This emerging class of enzymes now rivals heme enzymes in the diversity of oxidative transformations catalyzed.…”

Involvement of a third histidine in the ferrous active site of isopenicillin N synthase of Cephalosporium acremonium repudiated by recombinant double histidine mutants

“…From the ~H-NMR spectra of cIPNS, three imidazole groups were thought to ligate to Fe2 § and Ming et al,(4) postulated an iron coordination model in which three histidines were required for the formation of isopenicillin N. However, site-directed mutagenesis investigations of cIPNS revealed the involvement of only two histidine residues, H216 and H272, by the loss of IPNS activity upon replacement of these histidines individually with leucine (1,5). The removal of the imidazole group of these histidine residues with the concomitant loss of activity indicated that these side groups are involved in catalysis.…”

“…1) of penicillin and cephalosporin antibiotics. Alignment of the amino acid sequence of IPNS with other nonheme ferrous-requiring enzymes have shown that IPNS share three regions of relatively high sequence conservation (1). Since the involvement of conserved cysteine residues in iron binding has been discounted (2), histidine residues have been considered as potential ligands for iron binding (3).…”