Download MendeleySpectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase.
Rabe, P., Walla, C.C., Goodyear, N.K., Welsh, J., Southwart, R., Clifton, I., Linyard, J.D.S., Tumber, A., Claridge, T.D.W., Myers, W.K., Schofield, C.J.(2022) J Biol Chem 298: 102249-102249
- PubMed: 35835215
- DOI: https://doi.org/10.1016/j.jbc.2022.102249
- Primary Citation of Related Structures:
7POY, 7PSW - PubMed Abstract:
Isopenicillin N synthase (IPNS) catalyzes formation of the β-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O 2 )-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O 2 to the IPNS-Fe(II)-ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19 F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O 2 analog nitric oxide, using the less mobile α6 for comparison. 19 F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron-electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.
Organizational Affiliation:
Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, Oxford, United Kingdom. Electronic address: [email protected].
