title: Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution.
abstract: Shewanella oneidensis is an environmentally versatile Gram-negative gamma-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 A resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30-1.60 A resolution, thus providing a rare opportunity to dissect the structure-function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride-Meisenheimer complex.
authors.affiliation: Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Elegheert
authors.forename: Debbie
authors.initials: D
authors.lastname: van den Hemel
authors.forename: Ina
authors.initials: I
authors.lastname: Dix
authors.forename: Jan
authors.initials: J
authors.lastname: Stout
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Ann
authors.initials: A
authors.lastname: Brigé
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D009252:NADPH Dehydrogenase / Q000737:chemistry*; D020592:Shewanella / Q000201:enzymology
chemical_list: D009252:NADPH Dehydrogenase