The Structure of the Lipid A from the Halophilic Bacterium Spiribacter salinus M19-40T.

The study of the adaptation mechanisms that allow microorganisms to live and proliferate in an extreme habitat is a growing research field. Directly exposed to the external environment, lipopolysaccharides (LPS) from Gram-negative bacteria are of great appeal as they can present particular structural features that may aid the understanding of the adaptation processes. Moreover, through being involved in modulating the mammalian immune system response in a structure-dependent fashion, the elucidation of the LPS structure can also be seen as a fundamental step from a biomedical point of view. In this paper, the lipid A structure of the LPS from Spiribacter salinus M19-40T, a halophilic gamma-proteobacteria, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This revealed a mixture of mono- and bisphosphorylated penta- to tri-acylated species with the uncommon 2 + 3 symmetry and bearing an unusual 3-oxotetradecaonic acid.

[Peroxidase activity of octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus].

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s­1; k cat/K m = 855 mM­1 s­1) has been 100­300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Formation of a Subunit Form of the Core Light-Harvesting Complex from Sulfur Purple Bacteria Ectothiorhodospira haloalkaliphila with Different Carotenoid Composition.

B820 subunits from a purple sulfur bacterium Ectothiorhodospira. haloalkaliphila strain ATCC 51935T were obtained by treatment of Carotenoid free LH I-RC complexes of this bacterium with P--octylglu- copyranoside (ß-OG). The same complexes with 100% carotenoid content were unable to dissociate to B820 subunits, but disintegrated to monomeric bacteriochlorophyll (BChl) regardless of their carotenoid compo- sition. The degree of dissociation of the LH 1-RC complexes with an intermediate content of carotenoids (the' B820 formation) was directly dependent on the quantity of carotenoids in the samples. The resulting B820 subunits did not contain carotenoids. B820 subunits easily aggregated to form a complex with an absorption . peak at 880 nm at decreased ß-OG concentration. Analysis of the spectra of the LH I-RC complexes isolated from the cells with different'levels of carotenogenesis inhibition led to the conclusion of the heterogeneity of the samples with a predominance in them of (a) the fraction with 100% of carotenoids and (b) the fraction of carotenoid free complexes.

Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Šresolution) and sulfite (1.8 Šresolution), the structure of TvNiR in a complex with nitrite (1.83 Šresolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.