title: Active-site-mediated elimination of hydrogen fluoride from a fluorinated substrate analogue by isopenicillin N synthase.
abstract: Isopenicillin N synthase (IPNS) is a non-haem iron oxidase that catalyses the formation of bicyclic isopenicillin N from delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). In this study we report a novel activity for the iron of the IPNS active site, which behaves as a Lewis acid to catalyse the elimination of HF from the fluorinated substrate analogue, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-beta-fluorovaline (ACbetaFV). X-Ray crystallographic studies of IPNS crystals grown anaerobically with ACbetaFV reveal that the valinyl beta-fluorine is missing from the active site region, and suggest the presence of the unsaturated tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-isodehydrovaline in place of substrate ACbetaFV. (19)F NMR studies confirm the release of fluoride from ACbetaFV in the presence of the active IPNS enzyme. These results suggest a new mode of reactivity for the IPNS iron centre, a mechanism of action that has not previously been reported for any of the iron oxidase enzymes.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Annaleise R
authors.initials: AR
authors.lastname: Grummitt
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Rutledge
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: Jack E
authors.initials: JE
authors.lastname: Baldwin
mesh_terms: D000693:Anaerobiosis; D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray / Q000379:methods; D005459:Fluorides / Q000378:metabolism*; D006858:Hydrofluoric Acid / Q000378:metabolism*; D008961:Models, Structural; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D013379:Substrate Specificity; D014633:Valine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism
chemical_list: D009842:Oligopeptides; C011638:5-(2-aminoadipyl)cysteinylvaline; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D014633:Valine; D005459:Fluorides; D006858:Hydrofluoric Acid