title: 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.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: Wei
authors.initials: W
authors.lastname: Ge
authors.forename: Robert M
authors.initials: RM
authors.lastname: Adlington
authors.forename: Jack E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Rutledge
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008024:Ligands; D008738:Methyl Ethers / Q000737:chemistry; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010406:Penicillins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry*; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D008024:Ligands; D008738:Methyl Ethers; D010406:Penicillins; D047090:beta-Lactams; D014867:Water; D010088:Oxidoreductases; C006414:penicillin N; D010100:Oxygen
keywords: AC-; AC-O-methyl-d-allo-threonine; AC-O-methyl-d-threonine; AC-d-allo-isoleucine; AC-d-isoleucine; AC-d-thio-allo-isoleucine; AC-d-thioisoleucine; AC-d-valine; ACI; ACV; ACaI; ACmT; ACmaT; ACtI; ACtaI; Antibiotic; Biosynthesis; IPN; IPNS; Metalloenzyme; Non-heme iron oxidase; Penicillin; Protein crystallography; isopenicillin N; isopenicillin N synthase; δ-l-α-aminoadipoyl-l-cysteinyl-