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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.

SUBMITTER: Elegheert J

PROVIDER: S-EPMC2805545 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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

Elegheert Jonathan J van den Hemel Debbie D Dix Ina I Stout Jan J Van Beeumen Jozef J Brigé Ann A Savvides Savvas N SN

Acta crystallographica. Section F, Structural biology and crystallization communications 20091225 Pt 1

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) an ...[more]

PMID: 20057079

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Project description:Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering.

Dataset Information

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

Publications

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

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