Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies.
Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-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 P2(1)2(1)2(1), 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 P3(1)21, 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 +/- 0.003 nm.
Roach
P L
PL
Dyson Perrins Laboratory, University of Oxford, UK.
Clifton
I J
IJ
Hensgens
C M
CM
Shibata
N
N
Long
A J
AJ
Strange
R W
RW
Hasnain
S S
SS
Schofield
C J
CJ
Baldwin
J E
JE
Hajdu
J
J
D000693:Anaerobiosis; D001233:Aspergillus nidulans / Q000201:enzymology; D018360:Crystallography, X-Ray; D005296:Ferrous Compounds; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry; D010088:Oxidoreductases / Q000648:ultrastructure*; D011994:Recombinant Proteins; D013057:Spectrum Analysis
D005296:Ferrous Compounds; D008024:Ligands; D009842:Oligopeptides; D011994:Recombinant Proteins; D010088:Oxidoreductases; C038370:isopenicillin N synthetase