The reaction cycle of isopenicillin N synthase observed by X-ray diffraction

• Published in: Nature (London) Vol. 401; no. 6754; pp. 721 - 724
• Main Authors: Baldwin, Jack E, Burzlaff, Nicolai I, Rutledge, Peter J, Clifton, Ian J, Hensgens, Charles M. H, Pickford, Michael, Adlington, Robert M, Roach, Peter L
• Format: Journal Article
• Language: English
• Published: London Nature Publishing 14.10.1999; Nature Publishing Group
• Subjects: Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Biosynthesis; Chemical reactions; Crystallography; Crystallography, X-Ray; Crystals; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Interception; Iron; Ligases; Models, Molecular; Oligopeptides - metabolism; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Protein Conformation; X-ray diffraction; X-rays; Enzymatic activity; Molecular structure; Enzyme; Penicillin; Reaction mechanism; Biosynthesis; Synthase; Substrate enzyme complex; X ray diffraction; Thallophyta; Crystalline structure
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/44400

Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the biosynthesis of isopenicillin N (IPN), the precursor of all penicillins and cephalosporins. The key steps in this reaction are the two iron-dioxygen-mediated ring closures of the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered β-lactam ring forms initially, associated with a highly oxidized iron(IV)-oxo (ferryl) moiety, which subsequently mediates closure of the five-membered thiazolidine ring. Here we describe observation of the IPNS reaction in crystals by X-ray crystallography. IPNS·Fe2+·substrate crystals were grown anaerobically, exposed to high pressures of oxygen to promote reaction and frozen, and their structures were elucidated by X-ray diffraction. Using the natural substrate ACV, this resulted in the IPNS·Fe2+·IPN product complex. With the substrate analogue, δ-(L-α-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC) in the crystal, the reaction cycle was interrupted at the monocyclic stage. These mono- and bicyclic structures support our hypothesis of a two-stage reaction sequence leading to penicillin. Furthermore, the formation of a monocyclic sulphoxide product from ACmC is most simply explained by the interception of a high-valency iron-oxo species.