7POY

Spin labeled IPNS S55C variant in complex with Fe, ACV and NO

Summary for 7POY

• Entry DOI: 10.2210/pdb7poy/pdb
• Related: 6ZAM 6ZAN 6ZAP
• Descriptor: Isopenicillin N synthase, FE (III) ION, L-D-(A-AMINOADIPOYL)-L-CYSTEINYL-D-VALINE, ... (7 entities in total)
• Functional Keywords: isopenicillin n synthase, oxidase, penicillin biosynthesis, spin labeled protein, oxidoreductase
• Biological source: Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139)
• Total number of polymer chains: 1
• Total formula weight: 38613.71

Authors

Rabe, P.,Clifton, I.,Walla, C.,Schofield, C.J. (deposition date: 2021-09-10, release date: 2022-07-20, Last modification date: 2024-11-20)

Primary citation

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.
Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase.
J.Biol.Chem., 298:102249-102249, 2022

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)-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 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 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 analog nitric oxide, using the less mobile α6 for comparison. 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.

PubMed: 35835215
DOI: 10.1016/j.jbc.2022.102249

Experimental method

X-RAY DIFFRACTION (1.75 Å)