7psw
Spin labeled IPNS S55C variant in complex with Fe and ACV under anaerobic conditionsSpin labeled IPNS S55C variant in complex with Fe and ACV under anaerobic conditions
Structural highlights
Function[IPNS_EMENI] Removes, in the presence of oxygen, 4 hydrogen atoms from delta-L-(alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to form the azetidinone and thiazolidine rings of isopenicillin. Publication Abstract from PubMedIsopenicillin N synthase (IPNS) catalyses formation of the beta-lactam and thiazolidine rings of isopenicillin N (IPN) from its linear tripeptide L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) substrate in an iron and dioxygen (O2) dependent four electron oxidation without precedent in current synthetic chemistry. Recent X-ray free electron laser (XFEL) studies including time-resolved serial femtosecond crystallography show binding of O2 to the IPNS:Fe(II):ACV complex induces unexpected conformational changes in alpha-helices on the surface of IPNS, in particular in alpha3 and alpha10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed (19)F NMR and EPR experiments with labelled IPNS variants, we investigated motions in alpha3 and alpha10 induced by binding of ferrous iron, ACV and the O2 analogue NO, using the less mobile alpha6 for comparison. (19)F NMR studies were carried out on singly and doubly labelled alpha3, alpha6 and alpha10 variants at different temperatures. In addition, double electron-electron resonance (DEER) EPR analysis was carried out on doubly spin labelled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including alpha3 and alpha10 are induced by binding of ACV and NO. 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 non-heme oxygenase catalysis. Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase.,Rabe P, Walla CC, Goodyear NK, Welsh J, Southwart R, Clifton I, Linyard JDS, Tumber A, Claridge TDW, Myers WK, Schofield CJ J Biol Chem. 2022 Jul 11:102249. doi: 10.1016/j.jbc.2022.102249. PMID:35835215[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
|
| ||||||||||||||||||||