title: Analysis of glutamines in catalysis in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis.
abstract: Isopenicillin N synthase (IPNS), an important enzyme in the beta-lactam antibiotic biosynthetic pathway, is responsible for the catalytic conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Three catalytic ligands essential for IPNS activity have already been determined. Based on an Aspergillus nidulans IPNS crystal structure, the probable involvement of a fourth amino acid as a catalytic ligand was previously revealed. To continue the search for the fourth catalytic ligand, we report investigations on whether or not glutamines play a role in the catalytic action of Cephalosporium acremonium IPNS (cIPNS). Three glutamine residues were targeted for modification based on the previous revelation of one (Q337) via crystal structure coordinates, the conservation of one (Q234) in isozyme alignment and the proximity of one (Q227) to the catalytic centre. Analysis of the biotransformed mutant enzymes showed retention of activity, thereby rejecting the involvement of a possible glutamine as a catalytic ligand in cIPNS catalysis.
authors.affiliation: Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore.
authors.forename: P
authors.initials: P
authors.lastname: Loke
authors.forename: T S
authors.initials: TS
authors.lastname: Sim
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D008024:Ligands; D005973:Glutamine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase