The reaction cycle of isopenicillin N synthase observed by X-ray diffraction.
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 delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered beta-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 x Fe2+ x IPN product complex. With the substrate analogue, delta-(L-alpha-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.
Burzlaff
N I
NI
The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford, UK.
Rutledge
P J
PJ
Clifton
I J
IJ
Hensgens
C M
CM
Pickford
M
M
Adlington
R M
RM
Roach
P L
PL
Baldwin
J E
JE
D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D009842:Oligopeptides; C011638:5-(2-aminoadipyl)cysteinylvaline; D010088:Oxidoreductases; C038370:isopenicillin N synthetase