The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

Abstract

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the β-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.