Spectroscopic and kinetics studies of the inhibition of pig kidney diamine oxidase by anions

The role of copper in pig kidney diamine oxidase has been probed by examining the effects of potential Cu(II) ligands on the spectroscopic and catalytic properties of the enzyme. In the presence of azide and thiocyanate, new absorption bands are evident at 410 nm (epsilon = 6300 M-1 cm-1) and 365 nm (epsilon = 3000 M-1 cm-1), respectively. These bands are assigned as ligand-to-metal charge-transfer transitions, N3-/SCN- leads to Cu(II). One anion/Cu(II) is coordinated in an equitorial position. Anion binding can be completely reversed by dialysis. The equilibrium constants for diamine oxidase-anion complex formation are 134 M-1 (N3-) and 55 M-1 (SCN-). Azide and thiocyanate are linear uncompetitive inhibitors with respect to the amine substrate when O2 is present at saturating concentrations. Taken together, the data are consistent with a functional role for Cu(II) in diamine oxidase catalysis.     

Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.     

Effects of freshwater sponge Ephydatia fluviatilis on conjugative transfer of antimicrobial resistance in Enterococcus faecalis strains in aquatic environments

Filter feeding is a biotic process that brings waterborne bacteria in close contact with each other and may thus support the horizontal transfer of their antimicrobial resistance genes. This laboratory study investigated whether the freshwater sponge Ephydatia fluviatilis supported the transfer of vancomycin resistance between two Enterococcus faecalis strains that we previously demonstrated to exhibit pheromone responsive plasmid conjugation. Microcosm experiments exposed live and dead colonies of laboratory - grown sponges to a vancomycin-resistant donor strain and a rifampicin-resistant recipient strain of Ent. faecalis. Enterococci with both resistance phenotypes were detected on double selection plates. In comparison to controls, abundance of these presumed transconjugants increased significantly in water from sponge microcosms. Homogenized suspensions of sponge cells also yielded presumed transconjugants; however, there was no significant difference between samples from live or dead sponges. Fluorescent in situ hybridization analysis of the sponge cell matrix using species-specific probes revealed the presence of enterococci clusters with cells adjacent to each other. The results demonstrated that sponge colonies can support the horizontal transfer of antimicrobial resistance although the mechanism underlying this process, such as binding of the bacteria to the sponge collagen matrix, has yet to be fully elucidated.     

Copper-tyrosyl radical enzymes

Advances have been made since 2000 that contribute to our understanding of the biogenesis, structure and mechanism of copper-containing tyrosyl radical enzymes. Efforts to detail the biogenesis of galactose oxidase have produced the structure of the precursor enzyme, which provides a framework for emerging mechanistic studies. The role of the tyrosyl radical of cytochrome c oxidase is being defined in studies that aim to understand the His-Tyr crosslink, the location of the radical and, by direct attempts, to provide evidence for the radical during turnover.