Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction.
BACKGROUND
Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring.


RESULTS
Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed.


CONCLUSIONS
In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.
Ogle
J M
JM
The Dyson Perrins Laboratory, University of Oxford, UK.
Clifton
I J
IJ
Rutledge
P J
PJ
Elkins
J M
JM
Burzlaff
N I
NI
Adlington
R M
RM
Roach
P L
PL
Baldwin
J E
JE
D018360:Crystallography, X-Ray; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010088:Oxidoreductases; C038370:isopenicillin N synthetase