RESUMO
Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
Assuntos
Elétrons , Oxirredutases , Catálise , Domínio Catalítico , Cristalografia por Raios X , Compostos Férricos , Humanos , Lasers , Oxirredutases/química , Oxigênio/química , Penicilinas/química , Penicilinas/metabolismo , Especificidade por SubstratoRESUMO
An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
Assuntos
Dioxigenases/química , Ferroproteínas não Heme/química , Sítios de Ligação , Dioxigenases/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , Ligantes , Conformação Molecular , Ferroproteínas não Heme/metabolismoRESUMO
We report a Monte Carlo side chain entropy (MC-SCE) method that uses a physical energy function inclusive of long-range electrostatics and hydrophobic potential of mean force, coupled with both backbone variations and a backbone dependent side chain rotamer library, to describe protein conformational ensembles. Using the MC-SCE method in conjunction with backbone variability, we can reliably determine the side chain rotamer populations derived from both room temperature and cryogenically cooled X-ray crystallographic structures for CypA and H-Ras and NMR J-coupling constants for CypA, Eglin-C, and the DHFR product binary complexes E:THF and E:FOL. Furthermore, we obtain near perfect discrimination between a protein's native state ensemble and ensembles of misfolded structures for 55 different proteins, thereby generating far more competitive side chain packings for all of these proteins and their misfolded states.