title: Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium isopenicillin N synthase.
abstract: Isopenicillin N synthase (IPNS) is instrumental in the catalytic conversion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to a bioactive intermediate isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. It has recently been shown that this reaction is dependent on a conserved aspartate, D214, in a bacterial Streptomyces jumonjinensis IPNS. Thus, this study was carried out to provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremonium IPNS (cIPNS). Initially, alteration of the aspartate residue to generate the mutant D218L cIPNS protein was achieved by site-directed mutagenesis. Subsequent enzyme assays indicated that the catalytic property of the mutant protein was lost, attesting to the need for the corresponding conserved aspartate to maintain IPNS functionality. It is also evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consideration when site-directed mutant proteins are analysed for catalytic function.
authors.affiliation: Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore.
authors.forename: P
authors.initials: P
authors.lastname: Loke
authors.forename: J
authors.initials: J
authors.lastname: Sim
authors.forename: T S
authors.initials: TS
authors.lastname: Sim
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010406:Penicillins
chemical_list: D010406:Penicillins; D001224:Aspartic Acid; D010088:Oxidoreductases; C038370:isopenicillin N synthetase