Simultaneous functioning of different light-harvesting complexes-a strategy of adaptation of purple bacterium Rhodopseudomonas palustris to low illumination conditions.

Novel peripheral light-harvesting (LH) complex designated as LL LH2 was isolated along with LH4 complex from Rhodopseudomonas palustris cells grown under low light intensity (LL). FPLC-MS/MS allowed to reveal PucABd and PucBabc apoproteins in LL LH2 complex, which is different from previously described LH4 complex containing PucABd, PucABa and PucBb. The main carotenoids in LL LH2 complex were rhodopin and 3,4-didehydrorhodopin. Three-dimensional modeling demonstrated which amino acid residues of all the ß-subunits could interact with carotenoids (Car) and bacteriochlorophyll a (BChl a). Analysis of amino acid sequences of α-subunits of both LL complexes showed presence of different C-terminal motifs, IESSVNVG in αa subunit and IESSIKAV in αd subunit, in the same positions of C-termini, which could reflect different retention force of LL LH2 and LH4 on hydroxyl apatite, facilitating successful isolation of these complexes. Differences of these LL complexes in protein and carotenoid composition, in efficiency of energy transfer from Car to BChl a, which is two times lower in LL LH2 than in LH4, allow to assign it to a novel type of light-harvesting complex in Rhodopseudomonas palustris.

Expression of thermophilic two-domain laccase from Catenuloplanes japonicus in Escherichia coli and its activity against triarylmethane and azo dyes.

BACKGROUND: Two-domain laccases are copper-containing oxidases found in bacteria in the beginning of 2000ths. Two-domain laccases are known for their thermal stability, wide substrate specificity and, the most important of all, their resistance to so-called «strong inhibitors¼ of classical fungal laccases (azides, fluorides). Low redox potential was found to be specific for all the two-domain laccases, due to which these enzymes lost the researchers' interest as potentially applicable for various biotechnological purposes, such as bioremediation. Searching, obtaining and studying the properties of novel two-domain laccases will help to obtain an enzyme with high redox-potential allowing its practical application. METHODS: A gene encoding two-domain laccase was identified in Catenuloplanes japonicus genome, cloned and expressed in an Echerichia coli strain. The protein was purified to homogeneity by immobilized metal ion affinity chromatography. Its molecular properties were studied using electrophoresis in native and denaturing conditions. Physico-chemical properties, kinetic characteristics, substrate specificity and decolorization ability of laccase towards triphenylmethane dyes were measured spectrophotometrically. RESULTS: A novel two-domain recombinant laccase CjSL appeared to be a multimer with a subunit molecular mass of 37 kDa. It oxidized a wide range of phenolic substrates (ferulic acid, caffeic acid, hydroquinone, catechol, etc.) at alkaline pH, while oxidizing of non phenolic substrates (K4[Fe(CN)6], ABTS) was optimal at acidic pH. The UV-visible absorption spectrum of the purified enzyme was specific for all two-domain laccases with peak of absorption at 600 nm and shoulder at 340 nm. The pH optima of CjSL for oxidation of ABTS and 2, 6-DMP substrates were 3.6 and 9.2 respectively. The temperature optimum was 70 °C. The enzyme was most stable in neutral-alkaline conditions. CjSL retained 53% activity after pre-incubation at 90 °C for 60 min. The enzyme retained 26% activity even after 60 min of boiling. The effects of NaF, NaN3, NaCl, EDTA and 1,10-phenanthroline on enzymatic activity were investigated. Only 1,10-phenanthroline reduced laccase activity under both acidic and alkaline conditions. Laccase was able to decolorize triphenylmethane dyes and azo-dyes. ABTS and syringaldehyde were effective mediators for decolorization. The efficacy of dye decolorization depended on pH of the reaction medium.

Hup-Type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential.

Three-dimensional structures of six closely related hydrogenases from purple bacteria were modeled by combining the template-based and ab initio modeling approach. The results led to the conclusion that there should be a 4Fe3S cluster in the structure of these enzymes. Thus, these hydrogenases could draw interest for exploring their oxygen tolerance and practical applicability in hydrogen fuel cells. Analysis of the 4Fe3S cluster's microenvironment showed intragroup heterogeneity. A possible function of the C-terminal part of the small subunit in membrane binding is discussed. Comparison of the built models with existing hydrogenases of the same subgroup (membrane-bound oxygen-tolerant hydrogenases) was carried out. Analysis of intramolecular interactions in the large subunits showed statistically reliable differences in the number of hydrophobic interactions and ionic interactions. Molecular tunnels were mapped in the models and compared with structures from the PDB. Protein-protein docking showed that these enzymes could exchange electrons in an oligomeric state, which is important for oxygen-tolerant hydrogenases. Molecular docking with model electrode compounds showed mostly the same results as with hydrogenases from E. coli, H. marinus, R. eutropha, and S. enterica; some interesting results were shown in case of HupSL from Rba. sphaeroides and Rvi. gelatinosus.