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Related Experiment Videos

Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction.

J M Ogle1, I J Clifton, P J Rutledge

  • 1The Dyson Perrins Laboratory, University of Oxford, UK.

Chemistry & Biology
|January 5, 2002
PubMed
Summary
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Time-resolved crystallography revealed how Isopenicillin N synthase (IPNS) forms penicillin precursors. An oxygenated intermediate was trapped, showing a novel mechanism for beta-lactam ring closure in antibiotic biosynthesis.

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Isopenicillin N synthase (IPNS) is crucial for synthesizing penicillin and cephalosporin antibiotics.
  • IPNS utilizes iron(II) and molecular oxygen to convert a linear tripeptide into a bicyclic precursor.
  • The reaction mechanism involves beta-lactam ring formation and thiazolidine ring closure.

Purpose of the Study:

  • To elucidate the early stages of the IPNS catalytic mechanism.
  • To investigate the role of oxygen in the reaction pathway.
  • To understand the formation of the beta-lactam ring.

Main Methods:

  • Time-resolved crystallography was used to capture reaction intermediates.
  • An isosteric substrate analogue was designed to intercept the catalytic pathway.

Related Experiment Videos

  • High-pressure oxygen initiated the reaction in crystalline enzyme-substrate complexes.
  • Flash freezing trapped an oxygenated product bound to the iron center.

    • Main Results:

    • An oxygenated product, a thiocarboxylate, was trapped at the iron center.
    • The study observed turnover of a substrate analogue.
    • A mechanism for the formation of the thiocarboxylate product was proposed.

    Conclusions:

    • A hydroperoxide intermediate was proposed to attack a thioaldehyde species in the absence of the natural reaction partner.
    • This provides insight into events preceding beta-lactam closure in the IPNS reaction.
    • The findings enhance understanding of the enzyme's mechanism with its natural substrate.