Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution

Shewanella oneidensis is an environmentally versatile Gram-negative gamma-proteo-bacterium 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 angstrom 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 angstrom 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

Respiration and Growth of Shewanella oneidensis MR-1 Using Vanadate as the Sole Electron Acceptor

Shewanella oneidensis MR-1 is a free-living gram-negative γ-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex

Asymmetric bioreduction of activated carbon–carbon double bonds using Shewanella yellow enzyme (SYE-4) as novel enoate reductase

AbstractShewanella yellow enzyme (SYE-4), a novel recombinant enoate reductase, was screened against a variety of different substrates bearing an activated double bond, such as unsaturated cyclic ketones, diesters, and substituted imides. Dimethyl- and ethyl esters of 2-methylmaleic acid were selectively reduced to (R)-configured succinic acid derivatives and various N-substituted maleimides furnished the desired (R)-products in up to >99% enantiomeric excess. Naturally occurring (+)-carvone was selectively reduced to (−)-cis-dihydrocarvone and (−)-carvone was converted to the diastereomeric product, respectively. Overall SYE-4 proved to be a useful biocatalyst for the selective reduction of activated CC double bonds and complements the pool of synthetic valuable enoate reductases