Anaerobic Crystallisation of an Isopenicillin N Synthase · Fe(II) · Substrate Complex Demonstrated by X‐Ray Studies

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Abstract
Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, δ‐(L‐α‐aminoadipoyl)‐L‐cysteinyl‐D‐valine (Aad‐Cys‐Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging‐drop vapour‐diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X‐rays to at least 0.11‐nm resolution at the European Synchrotron Radiation Facility and belong to the space group P212121, with unit‐cell dimensions a= 4.68, b= 7.15, c= 10.10 nm. Their asymmetric unit contains a single IPNS. Fe(II). Aad‐Cys‐Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X‐rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3121, with unit‐cell dimensions a= 10.10, b= 10.10, c= 11.567 nm. Their asymmetric unit also contains a single IPNS. Fe(II). Aad‐Cys‐Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well‐ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X‐ray‐absorption fine‐structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe‐S(Aad‐Cys‐Val) bond length of 0.234±20.003 nm.