Structural and Mechanistic Basis for Anaerobic Ergothioneine Biosynthesis

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-alpha-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria

Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

Crystallographic, biochemical, and genetic studies reveal the mechanism of Rap protein phosphatase activity within the phosphorelay pathway leading to sporulation in Bacillus species

Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives

Crystal packing of the c(6)-type cytochrome OmcF from Geobacter sulfurreducens is mediated by an N-terminal Strep-tag II

The putative outer membrane c-type cytochrome OmcF from Geobacter sulfurreducens contains a single haem group and shows homology to soluble cytochromes c(6), a class of electron-transfer proteins that are typically found in cyanobacterial photosynthetic electron-transfer chains. OmcF was overexpressed heterologously in Escherichia coli as an N-terminal Strep-tag II fusion protein and isolated using streptactin-affinity chromatography followed by size-exclusion chromatography. The structure was solved by Fe SAD using data collected to a resolution of 1.86 A on a rotating copper-anode X-ray generator. In the crystal, packing interactions in one dimension were exclusively mediated through the Strep-tag II sequence. The tag and linker regions were in contact with three further monomers of OmcF, leading to a well defined electron-density map for this engineered and secondary-structure-free region of the molecule

Temperature measurement on MOEMS micromirror plates under illumination

The Fraunhofer Institute for Photonic Microsystems (IPMS) develops and fabricates MOEMS micro-mirror arrays for a variety of applications in image generation, wave-front correction and pulse shaping. In an effort to extent the application range, mirrors are being developed that withstand higher light intensities. The absorbed light generates heat. Being suspended on thin hinges, and isolated from the bulk by an air gap, the mirrors heat up. Their temperature can be significantly higher than that of their substrate. In this paper we describe an experiment carried out to verify simulations on the temperature within the mirror plates during irradiation. We created a structure out of electrically connected mirror plates forming a four-point electrical resistor, and calibrated the thermal coefficient of the resistor in a temperature chamber. We irradiated the resistor and calculated the mirror temperature. In the experiment, the temperature in the mirror plates increased by up to 180 °C. The mirrors did not show significant damage despite the high temperatures. Also, the experiment confirms the choice of heat transport mechanisms used in the simulations. The experiment was done on 48 ?m x 48 ?m mirrors suspended over a 5 ?m air gap, using a 355 nm solid-state laser (4 W, up to 500 W/cm²)