Crystallographic studies on the reaction of isopenicillin N synthase with an unsaturated substrate analogue.
Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.
Elkins
Jonathan M
JM
Dyson Perrins Laboratory, Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, UK OX1 3QY.
Rutledge
Peter J
PJ
Burzlaff
Nicolai I
NI
Clifton
Ian J
IJ
Adlington
Robert M
RM
Roach
Peter L
PL
Baldwin
Jack E
JE
D002384:Catalysis; D018360:Crystallography, X-Ray; D005296:Ferrous Compounds / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010406:Penicillins / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
D005296:Ferrous Compounds; D010406:Penicillins; C006507:vinylglycine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; C006414:penicillin N; D005998:Glycine