Báo cáo khoa học: Metabolism and Energetics pot

Metabolism and Energetics D1-1 Proteomic profiling of ‘Rhodospirillum rubrum’ under different nitrogen conditions T. T. Selao, S. Nordlund and A. Nore ´ n Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SWEDEN Biological nitrogen fixation is a vital process in the global nitrogen cycle, transforming dinitrogen in the atmosphere into ammonia. Being a very energy-demanding and oxygen-sensitive process, both expression and activity of the nitrogenase enzyme are tightly regu- lated. Rhodospirillum rubrum is a free-living, photosynthetic, purple non-sulfur nitrogen-fixing bacterium, which has been extensively used as a model organism for metabolic and regulatory studies regarding nitrogen fixation. In order to understand the metabolic effects when changing between different nitrogen sources, we have studied both the sol- uble and the chromatophore membrane-associated proteome of R. rubrum. Using a combination of electrophoretic techniques and mass spectrometry analysis, the differential expression and cellular localization of several proteins were studied in relation to nitrogen fixing, non-nitrogen fixing and nif-gene derepressing conditions. Acknowledgement: This work was supported by grants from the Carl Tryggers Foundation to AN, from the Swedish Research Council to SN and from Fundacao para a Ciencia e a Tecnologia (FCT) – Portugal, through the PhD fellowship SFRH/BD/23183/ 2005 to TTS. D1-2 The role of the AmtB1-GlnJ complex on the regulation of nitrogen fixation in the phototrophic bacterium Rhodospirillum rubrum P. F. Teixeira, H. Wang, A. Jonsson, A. Nore ´ n and S. Nordlund Stockholm University, Stockholm, SWEDEN Rhodospirillum rubrum, a photosynthetic purple free-living bacter- ium is capable of performing biological nitrogen fixation, in a reac- tion catalysed by the metalloenzyme nitrogenase, a complex constituted by dinitrogenase and dinitrogenase reductase. The activity of nitrogenase is regulated by post-translational reversible ADP-ribosylation of dinitrogenase reductase in response to exter- nal effects, the so-called switch-off effect, catalysed by DRAT (di- nitrogenase reductase ADP-ribosyl transferase) and DRAG (dinitrogenase reductase activating glycohydrolase). In R. rubrum, the complex regulation of nitrogen fixation and assimilation is coordinated by three members of the PII family of signalling pro- teins: GlnB, GlnJ and GlnK. We have recently shown that 2-oxo- glutarate is a fundamental signal for nitrogen fixation and assimilation, most likely transduced by the PII proteins that are known to bind 2-oxoglutarate and ATP. In this work we have investigated the specific interaction between the ammonium transporter AmtB1 and the signalling protein GlnJ, and showed that the complex is sensitive to the concentration of 2- oxoglutarate, in the presence of ATP and a divalent cation (Mg2+ or Mn2+). Also, the importance of this complex in the regulation of post- translational modification of nitrogenase (through the DRAT/ DRAG system) was also analyzed. D1-3 De novo biosynthesis of vitamin B6 – Complex formation of the heteromeric Bacillus subtilis PLP synthase M. Neuwirth, S. Wallner, K. Flicker and P. Macheroux Graz University of Technology, Graz, AUSTRIA Pyridoxal-5-phosphate (PLP), the biologically active form of vita- min B6, serves as an essential cofactor in many enzymatic reac- tions. In plants, fungi, archaea, protozoa and some eubacteria its de novo biosynthesis is accomplished via a recently discovered pathway different to the well-studied pathway of Escherichia coli. This major pathway leads to direct formation of PLP and is cata- lyzed by a PLP synthase complex. This heteromeric complex is made up from a synthase, termed Pdx1, and a glutaminase, termed Pdx2. Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1, which combines it with a triose- and a pentosephosphate sugar to form PLP. The interaction between Pdx1 and Pdx2 is essential for the catalytic activity of both domains. A characterization of the complex formation was carried out by isothermal titration calorimetry (ITC). Using this method, we have shown that L-glutamine stabilizes the complex leading 25-fold tigh- ter binding. Furthermore, L-glutamine determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions. Our current efforts focus on determination of the contribution of single amino acid residues to complex formation. Based on the 3D struc- ture (see companion abstract of Strohmeier et al.) amino acid resi- dues predicted to be important for the protein-protein interaction were exchanged and effects determined by ITC. D1-4 Vitamin B6 biosynthesis of Bacillus subtilis – uncovering secrets of a complex mechanism M. Strohmeier 1 , T. Raschle 2 , J. Mazurkiewicz 3 , K. Rippe 3 , I. Sinning 1 , T. B. Fitzpatrick 2 and I. Tews 1 1 Heidelberg University Biochemistry Center, Heidelberg, GERMANY, 2 ETH Zurich, Institute of Plant Sciences, Zu ¨ rich, SWITZERLAND, 3 Molecular Biophysics Group, Kirchhoff Institute for Physics, Heidelberg University, Heidelberg, GERMANY We determined, by x-ray crystallography, the 3D – structure of a $650 kDa bacterial glutamine amidotransferase, called pyridoxal 5-phosphate (PLP) synthase. Showing an overall cog-wheel like shape, the complex is composed of 12 glutaminase and 12 synthase subunits. One enzymatic unit is a synthase – glutaminase heterodi- mer, which is able to synthesise PLP, the biological active form of Vitamin B6 de novo. The glutaminase active site, only functional in the complex, comprises a catalytic triad to produce ammonia and glutamate from glutamine. By comparison of the free enzymes with those in the complex, we were able to describe a mechanism for glutaminase activation by protein – protein interaction 1 (see also companion paper of Neuwirth et al.). Ammonia must be transferred to the synthase active site, where from a pentasaccharide and a trisaccharide PLP gets formed. New data support a model for ammonia tunnelling by a transient tun- nel. Distinct cavities and constrictions for ammonia on its way through the enzyme were identified. Reference 1. Strohmeier M, Raschle T, Mazurkiewicz J, Rippe K, Sinning I, Fitzpatrick TB and Tews I PNAS. 103(51): 19284–19289 Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 221 D1-5 Heavy metal and mineral contents in yvgW mutant spores of Bacillus subtilis O ¨ . _ Irigu ¨ l, M. A. O ¨ ztu ¨ rk, N. Tokman, S. Akman and A. Karatas Istanbul Technical University, Istanbul, TURKEY Many gram-positive soil bacteria, such as Bacillus subtilis, produce dormant endospores which are resistant to a variety of physical and chemical treatment. In our previous studies, it was found that B.subtilis yvgW spores were resistant to lysozyme, but they were highly sensitive to heat and chloroform. The level of DPA was also significantly reduced to 63 % in yvgW spore. They were also shown to return to their vegetative state in a more delayed pattern. Although this defect was obviously observed in SMM minimal medium, no significant differences in outgrowth properties of yvgW null mutant and wild type spores were captured in 2xYT medium. All of these available data suggested that deficiency of yvgW might effect a late step in spore synthesis; spore coat/cortex synthesis, dehydration of spores, SASPs and DPA synthesis and/or accumulaton in the spores. On the other hand, along with the fact that yvgW gene was first reported as a membrane-metal efflux pro- tein, another rational possibility of YvgW to function in core min- eralization constructed the purpose of the present study. Under this respect,the residual metals as well as mineral contents in yvgW null mutant spores were measured by atomic absorption spectrom- etry and compared with wild type spores in order to gain more in- sights about the functional role of YvgW during sporulation. D1-6 Characterization of a -amylase and protease from thermostable bacteria isolated from hot spring (Diyadin, Turkey) F. Matpan and K. Gu ¨ ven Department of Biology University of Dicle, Diyarbakir, TURKEY In this study, water and mud samples were collected from Diyadin Hot Springs (Agri). The isolated thermostabile bacteria were char- acterized. Two of these thermostable, Gram positive, bacil and spore-forming isolates (K1 and D3) were chosen, and the produc- tion of exracellular enzymes a-amylase and protease was studied and these enzymes were then characterized. The production of ex- racellular a-amylase and protease in both isolates were studied in the period of 3–36 h. These enzymes were partially purified and then the optimum pH and temperature values for both enzymes were obtained. Moreover, the effects of heavy metal ions (Ca +2 ,Cu +2 ,Zn +2 , Mn +2 and Hg +2 ), PMSF, EDTA and SDS on the enzyme activity were tested. Finally, electrophoretic analysis of these enzymes were carried out. Keywords: a-amylase, enzyme characterization, enzyme inhibitors, hot spring, protease, thermophile bacteria D1-7 Purification and characterization of alpha-L-fucosidase from Paenibacillus thiaminolyticus E. Benesova, P. Lipovova, J. Riedl and B. Kralova ICT, Prague, CZECH REPUBLIC Alpha-L-fucosidase is an enzyme, which plays an important role in the metabolism of fucose-containing glycoconjugates of many organisms (bacteria, plants, mammals etc.). These exoglycosidase removes L-fucosyl residues from the non-reducing ends of sugar chains of oligosaccharides, glycoproteins and glycolipids, which are involved in many significant processes, such as cell-cell or ligand- receptor interactions, signal transduction or inflammation and growth regulation. As the fucose-containing compounds could find some applications in therapies of many severe diseases, e.g. cancer or in the treatment after transplatations, some fucosidases with the ability to catalyze transglycosylation reaction are under detailed investigation. In this work the bacterial strain Paenibacillus thiaminolyticus was determined as a producer of alpha-L-fucosidase. Basic characteris- tics were measured in the cytosolic extract. During the purification procedure it was discovered, that two isoenzymes (named fuciso1 and fuciso2) are present in this microorganism. Both isoenzymes were purified separately and their abilities to catalyze transglycosy- lation reaction were tested. The amino acid sequence of N-termi- nus of fuciso1 was determined. In the future work the possibility of immobilization of both isoen- zymes will be tested and sequences of genes, which encode fuciso1 and fuciso2, will be determined. D1-8 From structure to function: a novel cytochrome c nitrite reductase from extremophile bacteria Thioalkalivibrio nitratireducens A. Slutsky 1 , T. Tikhonova 1 , K. Boyko 1 , K. Polyakov 2 and V. Popov 1 1 Bach Institute of Biochemistry, Moscow, RUSSIAN FEDERATION, 2 Engelgardt Institute of Molecular Biology, Moscow, RUSSIAN FEDERATION A novel unique cytochrome c nitrite reductase (TvNiR) was puri- fied from the cell free extracts of the non-ammonifying obligately chemolithoautotrophic and haloalkaliphilic sulfur-oxidizing bac- teria Thioalkalivibrio nitratireducens. TvNiR shows no amino acid sequence homology with the known cytochrome c nitrite reductas- es. Three-dimensional structures of apo-TvNiR and the complexes with substrates have been determined with high resolution. TvNiR forms a highly symmetrical and stable hexameric structure result- ing in a unique ensemble of 48 hemes. In solution TvNiR exists also as a stable hexamer. The substrate specificity of TvNiR was characterized. The enzyme catalyzes reduction of nitrite with extre- mely high specific activity, hydroxylamine, sulfite and peroxide; possesses high thermostability and works under wide range of pH. Influence of inhibitors on the enzyme activity has been analysed. In spite of high nitrite reductase activity the fact that Tv. nitratire- ducens is incapable to grow with nitrite as an electron acceptor under anaerobic conditions allows us to hypothesize that TvNiR physiological function is not associated with nitrite respiration. The real physiological role of TvNiR and reasons of formation of so complicate catalytic machinery containing 48 haems is the sub- ject of our present investigation. Abstracts Metabolism and Energetics 222 ª 2007 The Authors Journal compilation ª 2007 FEBS D1-9 Structures of fatty acyl-(acyl carrier protei n) thioesters reveal a binding cavity that expands to fit longer substrates A. Roujeinikova 1 , W. J. Simon 2 , J. Gilroy 2 , D. W. Rice 3 , J. B. Rafferty 3 and A. R. Slabas 2 1 University of Manchester, Manchester, UK, 2 University of Durham, Durham, UK, 3 University of Sheffield, Sheffield, UK Acyl carrier protein plays a central role in de-novo fatty acid biosynthesis and a range of other metabolic events. We present crystallographic analysis of the structures of a family of acylated E. coli ACPs of varying acyl chain length. This study reveals that the growing acyl chain can be accommodated within a central cav- ity in the ACP for transport during the elongation stages of lipid synthesis through changes in the conformation of a four a-helix bundle. Our results not only clarify the means by which a substrate of varying size and complexity is transported in the cell but also suggest a mechanism by which interacting enzymes can recognize the loaded ACP through recognition of surface features including the conformation of the phosphopantetheine linker. D1-10 Cloning of two Bacillus anthracis arylamine N-acetyltransferases and evidence for the existence of a new NAT homolog devoid of NAT activity B. Pluvinage 1 , J. Dairou 1 , O. Mary-Possot 2 , M. Martins 1 , A. Fouet 2 , M. Mock 2 , J. Dupret 1 and F. Rodrigues-Lima 1 1 Universite Paris 7, Paris, FRANCE, 2 Institut Pasteur, Paris, FRANCE The arylamine N-acetyltransferases (NATs) are xenobiotic-metabo- lizing enzymes that catalyze the N-acetylation of arylamines, hy- drazines and their N-hydroxylated metabolites. We report here the cloning, functional expression and characterization of three new homologous NAT genes (referred as BaNAT1, BaNAT2 and BaNAT3) from the pathogen Bacillus anthracis. The corresponding proteins possess the characteristic NAT motifs, share around 40% sequence identity and were recognized by an anti-NAT antibody. However, these three NAT homologs were found to display dis- tinct structural and enzymic properties. BaNAT1 and BaNAT2 appeared to possess shorter C-terminal domains when compared to BaNAT3 and the other known NAT enzymes. Whereas BaNAT2 and BaNAT3 displayed significant NAT activity towards several prototypic NAT substrates (including two sulfonamide antibiotics SMX and SMZ in the case of BaNAT3), BaNAT2 was also found to possess acetyl-CoA (AcCoA) and p-nitrophenyl acetate (PNPA) hydrolysis activity indicating that this new bacterial NAT enzyme may also act as an AcCoA hydrolase in absence of arylamine sub- strates. BaNAT1, which is so far the shortest known NAT homo- log, was devoid of NAT or AcCoA/PNPA hydrolysis activities indicating that it is a new bacterial NAT-like protein with a yet unknown function. The three genes were endogenously expressed in B. anthracis and NAT activity was found in cell extracts. Our data indicate that B. anthracis is to date the first prokaryote expressing three NAT homologs with different functional properties. D1-11 Expression of the dehalogenase of Burkholderia sp. MBA4 is affected by iron avail ability Y. Faan and J. Tsang Department of Botany, University of Hong Kong, Pokfulam Road, Hong Kong, CHINA Burkholderia sp. MBA4 is able to utilize haloacids, such as mono- chloroacetate (MCA), as a sole source of carbon and energy. This ability is conferred by the production of a dehalogenase (DehIVa) which mediates the hydrolytic cleavage of the carbon-halogen bond. The expression of the DehIVa gene ( hdlIVa) is regulated and is repressed by the presence of pyruvate, succinate or branched-chain amino acids. In order to study the regulation of this gene, transposon mutagenesis was used. Plasmid pOT182-L, which carries a Tn5 derivative, was used to generate mutants with altered expression profile of hdlIVa. A reporter plasmid, pHKU1508, carrying a lacZ gene with a hdlIVa promoter, was electroporated into MBA4. Tn5 disrupted mutants with increased expression of hdlIVa were screened for increased degree of blue color development in medium containing pyruvate and X-gal. One of the mutants 15U07 shows a significant increase in the expression of hdlIVa when grown in medium containing pyruvate only or in medium containing both pyruvate and MCA. The disrupted gene in this mutant encodes protein containing a putative AfuA domain. This domain is the periplasmic component of an ABC- type Fe(III) ion transport system. A putative Ferric uptake regula- tor (FUR) binding site is present in the promoter of hdlIVa. Iron is probably transported into the cell by this transport system and interacts with Fur to repress the expression of hdlIVa. D1-12 Role of a tyrosine kinase and a phosphatase in exopolysaccharide synthesis by Burkholderia cepacia A. S. Ferreira, J. H. Leita ˜ o, S. A. Sousa, A. M. Cosme, I. Sa ´ -Correia and L. M. Moreira CEBQ, Instituto Superior Te ´ cnico, Lisbon, PORTUGAL Burkholderia cepacia complex bacteria are important opportunistic pathogens incystic fibrosis patients, able to lead to rapid decline of lung function, necrotizing pneumonia and septicaemia. About 80% of clinical isolates produce cepacian, an exopolysaccharide (EPS) that is hypothesized to be important in persistence and virulence of strains. Tyrosine phosphorylation and dephosphorylation by pro- tein tyrosine kinases (PTK) and phosphotyrosine phosphatases (PTP) are known to be important in post-translational modifica- tions of proteins that control EPS biosynthesis. In our work we are studding the role of BceD, a PTP, and BceF, a PTK, in cepa- cian production. The two proteins are encoded by genes belonging to bce gene cluster, responsible for cepacian production. Immuno- detection studies proved the presence of phosphorylated tyrosine residues on BceF and site directed mutagenesis on walkerA ATP binding motif showed that the protein is autophosphorylated. In vitro studies also proved BceD function as a PTP, able to dep- hosphorylate BceF. Disruption of bceF abolished cepacian produc- tion, but bceD mutant was still able to accomplish 75% of the production, although the EPS molecular weight was lower than the one produce by the parental strain. The size of in vitro biofilms produced by the two mutants is minor than the parental ones, but the biofilm size, as well as EPS production by the bceF mutant can be restore by complementation assays. Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 223 D1-13 Lipase super-induction system from Pseudomonas-like bacteria by fatty alcohols M. Ishizuka 1 , Y. Komatsu 1 , W. Tsukimura 1 and K. Ushio 2 1 Department of Applied Chemistry, Chuo University, Tokyo, JAPAN, 2 Niihama National College of Technology, Niihama, JAPAN Stable and high stereo-selective lipase and esterase have attracted much attention from viewpoints of industrial usage. In the course of our studies searching for efficient inducers for lipase, among various additives, we have found that fatty alcohols act as the most effective super-inducers for induction of lipase production by several Pseudomonas-like bacteria. The addition of fatty alcohols brought about more than several hundred-fold enhancement of the lipase activity compared to the case with no additive. This means several dozen-fold enhancement of lipase activity compared with olive oil grown case. Gram per litter lipase production has been capable. We also found that when several Pseudomonas-like bac- teria were grown on fatty alcohols, not only lipases but a large amount of an extracellular 15 kDa protein (P15) was strongly induced. This induction of P15 seems to be ‘simultaneous’ with that of lipases. P15 seems to be an activator-protein. Productions of those two proteins are possibly under the same regulation system. Under oxygen-limited condition, an extracellular 17 kDa protein was induced instead of P15. Expressed lipases, deduced from the cloned nucleotide sequences, have high homology among Pseudo- monas-like lipases including lactonizing lipase. Lactones can be syn- thesized from the corresponding x-hydroxy fatty acids, an inducer of cutinase in a fungal system, and above strains possess lactone- specific esterase. Although x-hydroxy palmitic acid did not induce ‘lipases’ from these bacteria, these lipases induced by fatty alcohols seem to be directed to cutine degradations. D1-14 Unveiling the unusual acylation behavior of type II fatty acid biosynthesis acyl carrier proteins A. Misra 1 , N. Surolia 2 and A. Surolia 1,3 1 Indian Institute of Science, Bangalore, INDIA, 2 Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, INDIA, 3 National Institute of Immunology, New Delhi, INDIA Acyl carrier protein (ACP) plays a central role in most metabolic processes inside the cell and almost 4% of the total enzymes inside the cell require it as a cofactor. Self-acylation of ACPs involved in polyketide biosynthesis pathways and absence of such an autocata- lytic process for ACPs involved in fatty acid biosynthesis has been noted. Here, we demonstrate self-acylation property in ACPs from P. falciparum and B. napus that are essential components of type II fatty acid biosynthesis (FAS), disapproving the existing notion that this phenomenon is restricted only to ACPs involved in poly- ketide biosynthesis. We also provide strong evidences to suggest that catalytic self-acylation is intrinsic to the individual acyl carrier protein. Mutational analysis of P. falciparum and B. napus acyl carrier proteins revealed the key residue(s) involved in this phe- nomenon. We also demonstrate that these ACPs involved in fatty acid synthesis exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mech- anism for the self-acylation reaction is also proposed. Further- more, we have also demonstrated that some type II FAS ACPs exhibit acyl-transferase behavior and self-acylation and acyl-transf- erase behavior are totally uncoupled. D1-15 Efficiency of commercial enzymatic preparations in grain hydrolysis for preparation of substrates for beer ferm entation J. Ivanovska, S. Mladenovic, B. Nastasijevic, S. Miletic and J. Acimovic University of Belgrade, Faculty of Chemistry, Belgrade, SERBIA The chemical composition of malt is of crucial importance for wort quality and together with conditions of fermentation process, they determine beer quality. Commercial enzymes preparations compen- sate enzymes naturally present in the malt. As substitutes for malt, originally made from malted barley, we used corn grits, rice and unmalted barley mixtures as adjunct for wort, and Ceremix and Termamil enzyme preparations (microbial origin). Wort quality was monitored by following parameters: filterability speed, a-amino-nitrogen, total sugars, and degree of saccharification. Beer quality is assessed, beside usual parameters, by presence and levels of highly volatile alcohol and esters, such as 3-methyl butanol, 1-propanol, phenyl ethanol and ethyl acetate that dictate flavour and taste of beer. Higher values of these substances were found in all samples of wort supplemented with enzymes, in comparison with those without adding of Termamyl and Ceremix. Although these preparations increase efficiency of saccarification process, their usage concomitantly with enzymes from malt leads to eleva- tion of substances that negatively contribute to beer flavour and taste. Addition of these enzyme preparations meets requests for good beer quality wherever enzymes from malt are not present in sufficient quantity. For optimal results 0.5% of Termamyl and Ceremix should be added, while in all other cases where malted substance is present with at least 50%, their usage should be strictly controlled. D1-16 Rsp5 is involved in co-regul ation of unsaturated fatty acid and ergosterol synthesis in yeast P. Kaliszewski 1 , A. Szkopinska 1 , T. Berges 2 and T. Zoladek 1 1 Institute of Biochemistry and Biophysics, PAS, Warszawa, POLAND, 2 Laboratoire de Genetique de la Levure, Universite de Poitiers, Poitiers, FRANCE Rsp5, yeast ubiquitin ligase, is an essential protein for regulation of unsaturated fatty acid synthesis by activation of Spt23 and Mga2 transcription activators. Recent data showed that Spt23 and Mga2 bind to genes which are required for sterol biosynthesis (Auld et al., 2006). Our studies indicate that rsp5 mutant strain shows decreased level of ergosterol and its intermediates down- stream from lanosterol in the pathway but the levels of early inter- mediates are unchanged. This suggests that Rsp5 may be responsible for regulation of levels of proteins which are involved in the middle of ergosterol pathway. Analysis of wt and rsp5 strains transformed with plasmids bearing versions of spt23Dtm and mga2Dtm which express protein product constitutively active revealed that the ergosterol level is increased. Thus, we concluded that Rsp5 regulates sterol level via Spt23 and Mga2 activation. We analyzed the level of ergosterol biosynthetic enzymes in strains overexpressing spt23Dtm, mga2Dtm or dominant negative allele rsp5DC. We observed increased levels of Idi1 in strains expressing spt23Dtm or mga2Dtm and decreased levels of Idi1 and Erg3 in strain overexpressing rsp5DC. These data indicate that IDI1 and ERG3 may be regulated by Spt23 and Mga2. Abstracts Metabolism and Energetics 224 ª 2007 The Authors Journal compilation ª 2007 FEBS D1-17 Ethanol productivity of Saccharomyces cerevisiae immobilized on polyhype polymer P. Karago ¨ z, E. Erhan, B. Keskinler and M. Ozkan Gebze Institute of Technology, Kocaeli, TURKEY Glucose consumption and ethanol production by Saccharomyces cerevisiae ATCC 26602 cells immobilized on polyhype polymeric material in stainless steel reactor whit a working volume of 101 ml was investigated. The reactor was operated at flow rates of 1, 2, 3, 6, 9 and 12 ml/min with 50 g/l glucose containing medium. Eth- anol productivity reached its highest value at a flow rate of 6 ml/ min. Reactor worked at highest efficiency when 50 g/l substrate was fed with a dilution rate of 1 ml/min. Examination of polymer pieces taken from the reactor with scanning electron microscope showed that yeast cells efficiently immobilized on the surface of polymer pieces. Rough surface structure of polymer was found to be very suitable for immobilization of yeasts. D1-18 Characterization and proteomic analysis of a polyunsaturated fatty acids producing fungus, Mortierella alpina S. Ho and F. Chen The University of Hong Kong, Hong Kong, CHINA Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (C20:4n-6, AA), are of increasing demand due to the beneficial effects on human health, and have been used in many food and medical researches. Mortierella alpina is one of the industrially important oleaginous fungi, especially for PUFAs production. A new strain of Mortierella alpina SC9 was isolated and identified through morphological analysis and molecular techniques, using internal transcribed spacer1 and 2 (ITS1, and ITS2), and 5.8S rDNA regions. During a 7-day fermentation, the PUFAs content of M. alpina SC9 varied between 189.83 and 240.00 mg/g. In order to enhance the industrial production of PUFAs, knowledge of fatty acids accumulation is crucial. Lipid droplet was considered as a neutral lipid storage organelle conventionally, but recently it has been considered as a dynamic organelle, which interacts with other organelles. In this study, a lipid droplet-enriched fraction of M. alpina SC9 was isolated, and the proteins of the fraction were identified through nano-LC-MS/MS analysis. This is the first research studying the lipid droplet-enriched fraction of this lower fungus with unsequenced genome. D1-19 FRET labelling of ATP synthase Z. Ristic 1 ,P.Vo ¨ ller 1 , H. Lill 1 , A. Visser 2 and D. Bald 1 1 Vrije Universiteit Amsterdam, Amsterdam, THE NETHERLANDS, 2 Wageningen University, Wageningen, THE NETHERLANDS ATP synthase is the main enzyme producing ATP, the currency of energy in living cells, from ADP and inorganic phosphate. The energy for this reaction is derived from a proton gradient across the bio-membrane in which ATP synthase is located. Energy con- version is performed in two steps: first the electrochemical energy of the proton gradient is converted into mechanical energy of sub- unit rotation, subsequently this mechanical energy is converted into chemical energy of ATP. 3–4 protons are translocated in order to synthesize 1 molecule ATP, suggesting an elastic energy storage mechanism. Fo ¨ rster resonance energy transfer (FRET) will be used to deter- mine structural changes of the enzyme involved in intermediate elastic energy storage. We introduced a variety of fluorophors into ATP synthase from E. coli and Bacillus PS3 for usage as FRET label. The efficiency of fluorescence labelling as well as function- ality of the ATP synthase complex will be discussed. D1-20 Effect of ferB mutation on the sensitivity of Paracoccus denitrificans to quinones V. Sedlacek 1 , I. Kucera 1 and R. J. M. van Spanning 2 1 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, CZECH REPUBLIC, 2 Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, Free University, Amsterdam, THE NETHERLANDS The FerB protein of Paracoccus denitrificans has previously been characterized as a NAD(P)H:Fe(III)chelate, quinone and chro- mate oxidoreductase. BLAST searching of the experimentally determined N-terminal sequence of FerB against the publicly accessible complete genome sequences of P. denitrificans 1222 provided the putative nucleotide sequence of the ferB gene. Based on this knowledge, the gene was interrupted by the suicide vector pRVS3. On testing for growth abilities in the presence of quinon- es, the ferB mutant was found to differ profoundly from the wild-type strain in that its specific growth rate was little influ- enced by as much as 5 lM 1,4-naphtoquinone (cf. 81 % inhibi- tion observed for the wild type). On the other hand, both strains responded comparably to ubiquinone-0, which acted inhibitory at concentrations above 10 lM. To resolve these differences, the mechanism of quinone reduction by FerB was investigated more thoroughly by examining oxygen consumption and cytochrome c reduction, the markers of a one-electron process. The results sug- gest that while p-benzoquinone derivatives are reduced directly to hydroquinones, reduction of naphtoquinones yields semiquinones, which in turn give rise to superoxide upon reaction with oxygen. This may constitute biochemical basis for the enhanced toxicity of naphtoquinones in the presence of FerB. Acknowledgement: This work was supported by the grants of the Grant Agency of the Czech Republic (525/07/106) and the Ministry of Education, Youth and Sports (MSM 0021622413). Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 225 D1-21 Unusually fast ligand dissociation from bacterial cytochrome bd: a clue to NO tolerance? V. B. Borisov 1 , E. Forte 2 , P. Sarti 2 , M. Brunori 2 , A. A. Konstantinov 1 and A. Giuffre ` 3 1 Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, RUSSIAN FEDERATION, 2 Department of Biochemical Sciences, University of Rome ’Sapienza’, Rome, ITALY, 3 Institute of Molecular Biology and Pathology, National Research Council of Italy, Rome, ITALY Cytochrome bd is a bacterial respiratory oxidase, not belonging to the superfamily of heme-copper oxidases. This oxidase is pref- erentially expressed under microaerobic conditions and in patho- genic bacteria seems to be required for adaptation to conditions created by host immunity. Escherichia coli cytochrome bd con- tains three hemes, b 558 , b 595 and d, but no copper. As a unique feature, this oxidase is stable in a single-electron reduced, ‘mixed valence’ MV state, with heme d 2+ bound to O 2 , NO or CO. Stopped-flow ligand-exchange experiments carried out at 20°C show that (i) O 2 dissociation from the MV enzyme proceeds at k = 78 ± 0.5/s; (ii) the NO dissociation rate from heme d 2+ is unusually high, being 0.133 ± 0.005/s for the fully reduced (R) enzyme and 0.036 ± 0.003/s for the MV state; and (iii) likewise, CO dissociates more rapidly from R (6.0 ± 0.2/s) than from MV cytochrome bd (4.20 ± 0.34/s). Ligand dissociation from heme d 2+ is thus considerably faster than for other hemeproteins and controlled by the redox state of the hemes b (presumably b 595 ). As NO is involved in the immune response and inhibits cyto- chrome bd, the rapid dissociation of NO from this enzyme may be relevant to patho-physiology of bacterial infection. D1-22 Cloning, and functional characterization of two NAT enzymes in the soil fungus Podospora anserina M. Martins 1 , P. Silar 2 , J. Dairou 1 , F. Malagnac 2 , F. Rodrigues-Lima 1 and J. M. Dupret 1 1 Laboratoire de Cytophysiologie et de Toxicologie cellulaire EA1553, Universite ´ Paris7, FRANCE, 2 Institut de Genetique et Microbiologie, Universite ´ Paris11, FRANCE Arylamine N-acetyltransferases (NAT) constitute a family of xeno- biotic-metabolizing enzymes (XME) that catalyse the transfer of an acetyl group from acetyl-coA (AcCoA) to aromatic amine and N-hydroxylated metabolites. We have identified and studied two homologous NAT genes from the filamentous ascomycetes soil fun- gus Podospora anserina referred here as PodoNAT1 and Podo- NAT2. Whereas PodoNAT2 encodes for a 34 kDa putative NAT enzyme, PodoNAT1 appears to encode for the largest NAT iso- form ever identified (38 kDa). To characterize these two new puta- tive NAT enzymes, the PodoNAT1 and PodoNAT2 genes were cloned and recombinantly expressed as N-terminal histidine-tagged proteins in E.coli. Purified proteins were found to cross-react with an anti-NAT antibody and to display NAT activity towards sev- eral prototypic NAT substrates including aniline compounds. This showed that PodoNAT1 and PodoNAT2 are functional NAT enzymes. Further kinetic analysis indicated that the PodoNAT2 isoform has much better catalytic efficiencies than PodoNAT1 (up to 10 times higher). In particular, PodoNAT2 was found to acety- late efficiently several aniline derivative pollutants such as 3,4-di- chloroaniline and 4-butoxyaniline (both are toxic pesticides intermediates). In vivo knock-out experiments and crystallization trials are currently being performed to further understand the func- tion of these enzymes. Our study will allow a better understanding of the role of NAT enzymes and paves the way to the potential utilization of such enzymes in bioremediation. D2-1 Characteristics of plasma membrane electron transport system in Leishmania donovani promastigotes UR6 T. Bera, Sr., D. Sudhahar, S. Biswas and R. Haque Division of Medicinal Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, INDIA Leishmania donovani promastigote, a parasitic protist, was demon- strated to be capable of reducing the four nonpermeable electron acceptors e.g., a-lipoic acid (ALA), 5, 5’-dithiobis (2-nitroaniline- N-sulphonic acid) (DTNS), 1, 2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide at the exoplasmic face of the plasma membrane. This transmembrane reduction of nonpermeable elec- tron acceptors ranging from -290 mV to +360 mV takes place at pH 6.4. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron accep- tors and oxygen. Inhibition observed with respect to the mitoch- ondrial electron transport inhibitors, ionophores and uncouplers on the reduction of ALA, DTNS, NQSA, ferricyanide and oxygen was not similar in most cases. But modifiers of sulfhydryl groups showed pronounced effects on these electron acceptor reduction. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b- cytochromes, cyanide insensitive oxygen redox side, Na + and K + channel, capsaicin inhibited energy coupling site and energy linked P-type ATPase.1’-Oxomenaquinone-7 appears to act as mobile electron carrier between the redox centers. Reduction of nonper- meable electron acceptors was partially inhibited by F 1 F 0 -ATPase inhibitors oligomycin, triethyltinbromide and DCCD. Isolated and purified plasma membrane from L. donovani promastigote showed preference for NADPH than NADH for the reduction of electron acceptors. This is also true for the plasma membrane mediated reduction of fumarate and pyruvate, which appears to be coupled with ATP synthesis. D2-2 Regulation of the NQR-operons expression in Vibrio harveyi and Klebsiella pneumoniae M. S. Fadeeva, E. A. Yakovtseva, Y. V. Bertsova and A. V. Bogachev Belozersky Institute, Moscow, RUSSIAN FEDERATION Some marine and pathogenic bacteria exploit unique enzyme called sodium-translocating NADH:quinone oxidoreductase (Na + -NQR) which couples electron transfer from NADH to quinone with transmembrane sodium ions translocation instead of expelling pro- tons as complex I does. For revealing physiological role of Na + - NQR the expression of genes encoding this enzyme was studied in marine bacterium Vibrio harveyi, and in enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl con- centration, value of pH and presence of an uncoupler in the growth media do not influence significantly the level of nqr expres- sion for both bacteria. In the same time nqr expression depends on growth substrates used by these bacteria. Especially Na + -NQR is highly repressed in V. harveyi at anaerobic growth of this microor- ganism and the nqr expression is modulated by electron acceptors and values of their redox potentials. Some features of this anaer- obic repression point out this process to be under the control of ArcAB regulatory system, but inactivation of acrB gene showed the latter effect was independent on ArcAB regulatory system. Abstracts Metabolism and Energetics 226 ª 2007 The Authors Journal compilation ª 2007 FEBS D2-3 Crystal structure of a new type of NADPH-dependent quinone oxidoreductase (QOR2) from Escherichia coli I. Kim 1 , H. Yim 1 , M. Kim 1 , D. Kim 1 , Y. Kim 1 , S. Cha 2 and S. Kang 1 1 School of Biological Sciences, Seoul National University, Seoul, REPUBLIC OF KOREA, 2 Beamline Division, Pohang Accelerator Laboratory, Pohang, REPUBLIC OF KOREA Escherichia coli QOR2 [NAD(P)H-dependent quinone oxido- reductase; a ytfG gene product], catalyzing two-electron reduction of methyl-1,4-benzoquinone, is a new type of quinone reducing enzyme with distinct primary sequence and oligomeric conforma- tion from previously known quinone oxidoreductases. The crystal structures of native QOR2 and QOR2-NADPH complex reveal that QOR2 consists of two domains (N-domain and C-domain) resembling that of NmrA, a negative transcriptional regulator, which is belonging to the short-chain dehydrogenase/reductase family. The N-domain adopting the Rossmann fold provides plat- form for the binding of NADPH, and the C-domain, containing a hydrophobic pocket connected to the NADPH-binding site, appears to play important roles in substrate binding. Asn143 near the NADPH-binding site has been identified to be involved in sub- strate binding and catalysis from structural and mutational analy- ses. Moreover, the remarkable decrease of several enzymes implicated in carbon metabolism of qor2-overexpressing strain and the growth retardation of the strain suggest that QOR2 could play some physiological roles beyond the quinone-reducing activity. D2-4 The effect of oligomerization on activity and thermostability in a bacterial quinone reductase A. Binter, S. Deller, S. Sollner and P. Macheroux Graz University of Technology, Graz, AUSTRIA YhdA, a thermostable NADPH:FMN oxidoreductase from Bacil- lus subtilis, which shows quinone reductase activity, occurs as a tetramer. The two extended dimer surfaces are packed against each other by a 90° rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137. To investigate the importance of those ion pair contacts, the K109L and D137 single as well as the K109L/D137L double mutant protein were generated, expressed and purified. These mutants were characterized with respect to ol- igomerization, showing that all the muteins form dimers in solu- tion. Consequently, the salt bridges are essential for stabilizing the dimer-dimer interface. The quinone reductase activity is drastically reduced compared to wild type YhdA. Detailed analysis of the two half reactions revealed that the reduction of quinones is not affec- ted whereas reduction of the bound FMN cofactor by NADPH is severely compromised. Thus, it appears that binding of NADPH to the dimeric enzyme is disturbed. Furthermore, we report a detailed study on the thermostability of the YhdA variants. Ther- mal unfolding of wild type YhdA and above-mentioned muteins was monitored and melting temperatures were determined by CD spectroscopy as well as differential scanning calorimetry. D2-5 Redox state of endogenous coenzyme Q regulates the inhibition of uncoupling protein activity in A. castellanii mitochondria A. K. Swida and W. Jarmuszkiewicz Adam Mickiewicz University, Poznan, POLAND The Acanthamoeba castellanii mitochondria contain an uncoupling protein (AcUCP) which dissipates the proton electrochemical gra- dient built up by respiratory chain. In isolated, phosphorylating A. castellanii mitochondria the proton re-uptake mediated by free fatty acid-activated AcUCP is inhibited by purine nucleotide (GTP) only when quinone (Q) is sufficiently oxidized [1]. In isola- ted A. castellanii mitochondria respiring in state 4 with external NADH or succinate, the linoleic acid-induced uncoupling protein activity is not affected by purine nucleotide (GDP) when Q redu- cing pathway is uninhibited. The progressive decrease of state 4 respiration by limitation of respiratory substrates availability leads to a full inhibitory effect of GDP. As the inhibitory effect of GDP is observed only when the Q-reducing pathway is decreased, we conclude that the inhibition of free fatty activated AcUCP by pur- ine nucleotides depends on coenzyme Q redox state not only in phosphorylating mitochondria, but also in non-phosphorylating respiration. This indicates that Q redox state could be a metabolic sensor for purine nucleotide inhibition of FFA-activated UCP, uni- versal for both: resting and phosphorylating respiration. Acknowledgement: This work was supported by grants from the Polish agencies: The Ministry of Scientific Research and AMU Faculty of Biology (PBWB 701/2006). D2-6 Lot6p, a quinone reductase involved in apoptosis S. Sollner 1 , M. Durchschlag 2 , A. Prem 1 , S. Deller 1 and P. Macheroux 1 1 Graz University of Technology, Graz, AUSTRIA, 2 Karl Franzens University Graz, Graz, AUSTRIA NAD(P)H:quinone reductases are flavoenzymes expressed in the cytoplasm and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. A well known example of those enzymes is human NQO1, which is primarily found in tumor tissues and was recently shown to be associated with the 20S proteasome. Here, we report identification of Lot6p, the first soluble quinone reductase from baker’s yeast. Localisation studies demonstrate accumulation of Lot6p in the cytosol. Despite sharing only 23% similarity to NQO1, Lot6p pos- sesses similar biochemical properties, i.e. it catalyzes the reduction of quinone substrates at the expense of NAD(P)H via a ping pong bi mechanism. In vivo experiments confirm that the enzymatic activity of Lot6p is consistent with the phenotype of both Lot6p knock-out and Lot6p overexpression strains. Moreover, we show that yeast cells lacking Lot6p display deficiency in the apoptosis process and, accordingly, Lot6p overexpression leads to a highly increased amount of apoptotic cells. Additionally, we demonstrate the association of Lot6p with the 20S proteasome and show that this interaction strongly depends on the presence and redox state of the flavin cofactor. Thus, our findings suggest that Lot6p plays a key role in regulation of apoptosis, presumably connected to its association with the 20S proteasome. Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 227 D2-7 Analysis of the molecular pathomechanism of two truncation mutations in the mito chondrial gene encoding cytochrome c oxidase subunit I H. T. Hornig-Do, S. Eckertz, K. Lanz and R. J. Wiesner Institute of Vegetative Physiology, University of Ko ¨ ln, Ko ¨ ln, GERMANY Mutations of mitochondrial DNA cause a widely diverse pattern of disorders in humans. To clarify why the A7339G and the G6930A ((D’Aurelio et al., 2001) point-mutation lead to com- pletely different clinical effects, although affecting the same gene (COI) and resulting in truncation of subunit I of cytochrome c oxidase (COXI), we have studied their pathomechanism in hybrids cells containing the patients mitochondria at various levels. Both truncated COXI proteins do not accumulate to steady state levels high enough to be detectable on Western blots, and only radioactive pulse experiments showed that they are synthesized at all, but degraded rapidly by inner membrane quality control pro- teases. Cytochrome c oxidase subunit II and IV were reduced pro- portionally to COX I, indicating impairment in complex assembly. Compensatory up-regulation of transcription, of mRNA stabiliza- tion, and of selective degradation of mutated mRNAs were not observed in both mutations. However, more than 90 % of mutated mtDNA copies encoding the short truncation (A7339G) are neces- sary to cause mitochondrial dysfunction, while <60 % of copies encoding the large truncation (G6930A) are sufficient. These data suggest a dominant effect on complex assembly of minute amounts of the large truncation protein, while the short truncation is recessive. D2-8 Arginine 274 and histidines 224 and 228 in complex I NuoCD subunit are important for the terminal electron transfer step L. L. Euro, G. Belevich, M. Wikstro ¨ m and M. Verkhovskaya Helsinki University, Helsinki, FINLAND NADH:ubiquinone oxidoreductase (complex I) is the entry point for electrons into respiratory chain of mitochondria and many bac- teria. It works as a proton pump coupling one electron transfer with translocation of two protons across the membrane. The molecular mechanism of energy conversion is not known, but it is generally accepted that the N2 cluster and quinone are the key participants in the terminal electron transfer step. Therefore, the protein surroundings of the N2 cluster and the quinone-binding site are of particular interest in investigation of complex I. We studied the effects of substitution for alanine of conserved histi- dines 224 and 228, located near the proposed quinone-binding site, and the arginine 274, resided close to the N2 cluster, in the subunit NuoCD of E. coli complex I. Our EPR studies showed that only R274A mutation resulted in shift of the midpoint redox potentials of the N2 cluster and another fast relaxing iron-sulfur cluster, ten- tatively assigned as N6b, to more negative values. The EPR spec- tra of purified complex I from H224A and H228A mutants did not differ from the wild type. We showed that none of the mutations significantly alter the quinone-binding site or the proton pumping machinery. Comparison with the results of corresponding muta- tions in Complex I from Y. lipolytica is discussed. D2-9 The O(H) to E(H) transition in cytochrome c oxidase: kinetics and thermodynamics of the catalytic act D. A. Bloch, I. Belevich, N. Belevich, M. I. Verkhovsky and M. Wikstro ¨ m University of Helsinki, Helsinki, FINLAND Proton translocation in the catalytic cycle of cytochrome c oxidase proceeds sequentially in a four-stroke manner: every electron dona- ted by cytochrome c drives the enzyme from one of four intermedi- ates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another pro- ton for production of water in the catalytic site. The correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques, after laser-acti- vated electron injection into the Paracoccus denitrificans enzyme. The extent of electric potential generation during initial electron equilibration between Cu A and heme a confirms that this reaction is not, whereas oxidation of heme a, is kinetically linked to the main proton translocation events. The observed kinetics establish the reaction sequence of the proton pump mechanism. The 10 us electron transfer to heme a raises the pKa of a ‘pump site’, which is loaded by a proton from the inside of the membrane in 150 us. This increases the redox potentials of both hemes a and a 3 , which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a3/Cu B center, and the electron is transferred to Cu B . Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane. We discuss the results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase. D2-10 Proton transfer in cytochrome c oxidase. ATR-FTIR approach E. Gorbikova, M. Wikstrom and M. I. Verkhovsky University of Helsinki, Helsinki, FINLAND Cytochrome c oxidase (CcO) is the terminal complex of respiratory chain of mitochondria and some aerobic bacteria. CcO accepts four electrons from cytochrome c that are consecutively transferred through the line of redox centers: Cu A , heme a, heme a 3 and Cu B . The electron transfer is coupled to translocation of four protons (‘pumped’ protons) across the membrane; four more protons (‘chemical’ protons) are taken up by the enzyme for oxygen reduc- tion chemistry. The efficient technique to resolve proton transfer is ATR-FTIR spectroscopy. We applied ATR-FTIR in combination with electrochemistry and visible spectroscopy to perform redox ti- tration of cytochrome c oxidase. From the titration we estimated equilibrium redox potentials (E m ) of all four redox centers, redox interaction and constants of electron flow. pH-dependence of E m s results in uptake of two ‘chemical’ protons upon CcO reduction. The approach allowed assign features in Reduced-minus-Oxidized (R-minus-O) FTIR spectra to each of four redox centers and may be used for identification of IR bands of other redox-active enzymes. One of the acceptors of the ‘chemical’ protons may be OH - group located near Cu B . Appearance of a ‘free’ OH group of structured water molecule was found in ‘water IR region’ upon the enzyme reduction. Acceptor of the second ‘chemical’ proton may be hydroxyl residue that signature of protonation was found. The pH-titration of the R-minus-O FTIR spectra revealed two pK val- ues 6.6 and 8.4 that are explained in terms of non-specific protons that are used for redox potential adjustment. Equilibrium states may be and most likely are different from transient ones that appear during catalytic cycle turnover. Time-resolved FTIR would be ideal to catch protonation events in real-time catalysis. Abstracts Metabolism and Energetics 228 ª 2007 The Authors Journal compilation ª 2007 FEBS D2-11 Novel haem c binding in a bacterial cytochrome requires a dedicated maturation system S. Hartshorne 1 , M. Kern 2 , B. Meyer 2 , T. A. Clarke 1 , M. Karas 2 , D. J. Richardson 1 and J. Simon 2 1 University of East Anglia, Norwich, UK, 2 Johann Wolfgang Goethe University, Frankfurt am Main, GERMANY In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c binding motif. A novel octa-haem c protein, MccA, was identified that con- tains only seven conventional haem c binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH sig- nature. Mass spectrometric analysis of purified MccA from Woli- nella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX 15 CH sequence involved in haem c binding. Spectroscopic characterization of MccA identi- fied an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the biosynthesis of MccA in W. succinogenes suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c binding motifs in bacterial proteomes. D2-12 Structural characterization of oxygen pathways in cytochrome ba 3 oxidase V. M. Luna, J. A. Fee and C. D. Stout The Scripps Research Institute, La Jolla, CA, USA Cytochrome ba 3 oxidase (ba 3 ) is a cytochrome c oxidase from the thermophilic bacterium Thermus thermophilus. As such, it catalyz- es the flow of electrons from reduced cytochrome c to dioxygen forming water concomitant with proton translocation. This pro- cess is essential to aerobic life. Dioxygen reduction takes place at the cytochrome a 3 -Cu B center of ba 3 . However, access from the outside of ba 3 to this enzymatic center is not yet fully understood. Here we present our efforts in structurally characterizing the oxy- gen pathways in ba 3 . We have computationally described the loca- tions and volumes of hydrophobic cavities first observed in previous crystal structures of ba 3 . To experimentally validate these hydrophobic cavities, we have also solved structures of ba 3 crys- tals that were placed under different pressurization conditions of xenon and krypton gas. We clearly observe xenon/krypton bind- ing pockets that track the hypothesized dioxygen pathway directly to the enzymatic center. This Y-shaped channel is unique among cytochrome c oxidases and offers several possibilities for dioxygen access. These structural studies are an excellent springboard for further studies of dioxygen migration in ba 3 . D2-13 Distribution of mitochondrial haplogroups in coronary artery disease F. Zimmermann 1 , O. Stanger 2 ,E.Mu ¨ ller 1 , M. Wiesbauer 1 , J. A. Mayr 1 , B. Paulweber 3 , B. Iglseder 4 , W. Renner 5 , W. Eder 1 , W. Sperl 1 and B. Kofler 1 1 Department of Pediatrics, University Hospital, Salzburg, AUSTRIA, 2 Department of Cardiac Surgery, University Hospital, Salzburg, AUSTRIA, 3 Department of Internal Medicine, University Hospital, Salzburg, AUSTRIA, 4 Department of Geriatrics, University Hospital, Salzburg, AUSTRIA, 5 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University, Graz, AUSTRIA Evident exist that oxidative stress plays a significant role in the pathogenesis of coronary artery disease (CAD). Mitochondria are one of the major sources of reactive oxygen species under physiolo- gical conditions. According to the role of mitochondria in oxida- tive phosphorylation and oxidative stress, an interaction between susceptibility to metabolic syndromes and mtDNA variability can be hypothesized. In this study the potential association of mtDNA haplogroups with the development of CAD was investigated. We included individuals with angiographically documented CAD from two university hospitals in Austria (Salzburg, Graz). Patients underwent multiplex primer extension analysis for the determin- ation of major European haplogroups. Haplogroup T was signifi- cantly prevalent in patients with CAD compared to controls (12.7% Salzburg n = 354; 15.6% Graz n = 179 vs. 8% n = 1632 P < 0.005). After adjustment for age, sex, body mass index, HDL-cholesterin, C-reactive protein and current smoking, hap- logroup T remained significantly associated with CAD [odds ratio 2.60 (95% CI 1.39–4.88), P = 0.003]. Our data suggest that mtDNA haplogroup T is a risk factor for the development of CAD in Middle Europe populations. D2-14 Decrease of aerobic mitochondrial energy metabolism in neuroblastoma R. G. Feichtinger 1 , J. A. Mayr 1 , N. Jones 1 , F. H. Schilling 2 , P. Kogner 3 , W. Sperl 1 and B. Kofler 1 1 University Hospital Salzburg, Paracelsus Medical University, Salz- burg, AUSTRIA, 2 Olga Hospital, Stuttgart, GERMANY, 3 Child- hood Cancer Research Unit, Karolinska, SWEDEN Neuroblastomas (NB) are the most frequent extra-cranial solid tumors in children, originating from the neural crest. A shift in cel- lular energy production from oxidative phosphorylation (OXPHOS) to anaerobic glycolysis, called Warburg effect, is a fundamental property of cancer. Association of proteins of the mitochondrial energy metabolism with carcinogenesis has been demonstrated for succinate dehydrogenase (complex II) in pheoch- romocytoma, another tumor of the neural crest. The aim of the present study was to determine specific alterations of OXPHOS in human NB tissues (n = 15) by measurement of the enzymatic activity of citrate synthase, complex I, complex II, complex III, COX and ATP synthase. Compared to non malignant tissues the activity of citrate synthase, a pace-maker enzyme of the Krebs cycle, was not altered in NB. Correlated to citrate synthase activity we found a residual activity of complex I (22%), complex II (7%), complex III (69%), COX (27%) and ATP synthase (19%) in NB tissues. The reduction of the activity of all complexes of the respiratory chain in NB indicates that in these tumors loss of respiration is not related to a defect of a single enzyme as shown for hereditary pheochromocytomas. Thus, an upstream effector, controlling the overall respiration, seems to cause the downregulation of the OXPHOS in NB. Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 229 D2-15 The regulation of the potassium transport by calcium J. Zielinska and A. Szewczyk Nencki Institute of Experimental Biology, Warsaw, POLAND Potassium transport through mitochondrial inner membrane was identified to trigger cytoprotection. This transport is strictly ion channel dependent and is similar to plasma membrane ion channels activity. In the inner mitochondrial membrane were found three dif- ferent potassium selective ion channels: ATP-regulated potassium (mitoK ATP ) channel, large conductance Ca 2+ -activated potassium (mitoBK Ca ) channel and voltage dependent potassium (mitoKv1.3) channel. MitoBK Ca channel was first found in human glioma cell line LN229. Later its presence was describe also in guinea pig ven- tricular cells, rat brain and rat skeletal muscle. Activity of this channel depends on mitochondrial membrane potential and calcium ions. This channel is activated by NS1619 and blocked by iberio- toxin, charibdotoxin or paxiline. Potassium flux caused by opening of the mitoBK Ca leads to changes in the respiration rate. Therefore respiration changes can indicate if the channel is opened or closed. We have shown that calcium in micromolar concentration range can increase respiration rate in isolated rat skeletal muscle mito- chondria. This increase is inhibited by nanomolar concentration of iberiotoxin, but not by cyclosporine A, a known the permeability transition pore inhibitor. Additional we discuss the possibility of regulation of mitochondrial potassium channels by various protein kinases. There are many observations suggesting modulation of mitochondrial potassium channels by cAMP-dependent protein kin- ases, different protein kinase C isoforms and protein kinase G. We are focused on establishing the role of mitochondrial potassium channel phosphorylation event in regulation of the channel. Also presence of different PKC isozymes in mitochondria fraction was verified. D2-16 Effect of mutant P450 oxidoreductase on P450 aromatase activity P. Kempna ´ 1 , A. Pandey 2 , P. Mullis 1 , G. Hofer 1 and C. Flu ¨ ck 1 1 Department of Pediatric Endocrinology, University of Bern, Bern, SWITZERLAND, 2 T. Kocher Institute, University of Bern, Bern, SWITZERLAND POR is essential flavoprotein involved in electron transfer to all microsomal P450 enzymes. We described mutations in human POR which cause a broad spectrum of diseases. In first study effect of POR mutations on activities of P450c17 (17a-hydroxylase; 17-, 20-lyase), a key enzyme of steroidogenesis, was assayed. Now we analysed the impact of POR mutants on activity of another ster- oidogenic enzyme, aromatase. Aromatase and POR proteins were co-expressed in yeasts and aromatase activity was measured in iso- lated microsomes. Mutations R454H and V489E, located in FAD domain lead to loss of aromatase and P450c17 activities. Mutation A284P of POR did not affect aromatase despite of inhibited p450c17, suggesting that A284 is involved in POR/P450c17 but not in POR/aromatase interaction. Mutations C566Y and V605F in NADPH-domain affected activities of both enzymes and aroma- tase reaction was inhibited stronger. Aromatase activity supported by C566Y and V605F POR mutants was more sensitive to NADPH amounts than wild and A284P POR. Substitution of co- factor by NADH abolished aromatase activity in presence of both NADPH-domain POR mutants and decreased activities of POR wt and POR A284P partially. We show that some POR mutations influence activity of aromatase differently than activity of P450c17. Mutations in NADPH-binding domain cause partial loss of activity, but appear to be more sensitive to NADPH shortage. As POR sup- ports all microsomal P450 enzymes, we suggest that variable effects of P450 mutations and polymorphisms on different P450 enzymes may explain the broad clinical spectrum of POR deficiency. D2-17 NADPH utilization of chorismate synthase and its evolutionary implications for the shikimate pathway H. Ehammer 1 , G. Rauch 1 , B. Kappes 2 and P. Macheroux 1 1 Institute of Biochemistry, Graz, AUSTRIA, 2 Department of Parasitology, Universita ¨ tsklinikum, Heidelberg, GERMANY Chorismate synthase (CS) is the seventh enzyme of the shikimate pathway which catalyzes the conversion of 5-enolpyruvylshikimate- 3-phosphate to chorismate, the central precursor for many aroma- tic compounds including the aromatic amino acids. Chorismate synthases require reduced flavin, which is not consumed during turnover. The generation of the reduced cofactor and its sequestra- tion by chorismate synthase allows the classification into monofunctional and bifunctional enzymes. The bifunctional CS from fungi and unicellular algae possess an intrinsic NADPH:FMN oxidoreductase activity. However, bacterial and plant enzymes require an external source of reduced flavin. In this study we introduce an in-vivo screening procedure that allows ana- lyzing for mono-/bi-functionality. A CS-deficient yeast Saccharo- myces cerevisae strain harbouring a plasmid with the gene for a CS is tested for growth on minimal medium lacking aromatic amino acids. Bifunctional enzymes could restore prototrophy whereas monofunctional enzymes were dependent on the coexpression of a bacterial oxidoreductase. This analysis revealed that bifunctionality is also present in the CS of protozoan species such as Tetrahymena thermophila and Toxoplasma gondii and are not restricted to fungi. In contrast, all bacterial -including cyanobacterial – and plant enzymes tested were found to be monofunctional. D2-18 Molecular crowding and photosynthetic energy transformation S. Haferkamp 1 , W. Haase 2 , H. van Amerongen 3 , C. W. Mullineaux 4 and H. Kirchhoff 1 1 WWU, Muenster, GERMANY, 2 MPI of Biophysics, Frankfurt a. M, GERMANY, 3 Wageningen University, Wageningen, THE NETHERLANDS, 4 Queen Mary University of London, London, UK Bioenergetic membranes are densely packed with integral protein complexes. In particular this holds for the most abundant mem- brane on earth, the thylakoid system of higher plants ($70% of the membrane area are proteins). Although it is expected that this extreme packing of proteins causes serious problems for membrane functions the functional significance of molecular crowding for energy transformation is poorly understood. We developed a pro- cedure which enables a controlled dilution of the protein density in isolated thylakoid fragments (grana thylakoids) by fusion with uni- lamellar lipid-liposomes. The expected dilution of the protein den- sity was confirmed by biochemical and ultrastructural analysis of the fusion products. A functional (spectroscopic) analysis of the diluted membranes shows a breakdown of an extended supramo- lecular protein network between photosystem II and light harvest- ing complexes II necessary for efficient collecting of light quanta by the photosynthetic machinery. This highlights the requirement of high protein packing for light harvesting in plants. We postulate that molecular crowding in grana membranes seems to ensure an effective energy transfer in a membrane with weakly interacting antenna complexes. The interplay between weak and strong inter- actions could reflect a compromise between a flexibility of the pro- tein arrangement and a high efficiency of light utilization. Abstracts Metabolism and Energetics 230 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... ligand interaction scan and suggest that the method may be applied to any protein given an appropriate activity assay Ligand-regulated mutants may then be expressed in cells, wherein they can be regulated by ligand administration Because of its simplicity and generality the ligand interaction scan method may complement other genetic and chemical genetic methods for analysis of protein function and. .. spectroscopy The obtained data and in silico docking studies gave further insights into possible modes of protein-ligand interactions and helped to understand the molecular basis of the observed biological effects ª 2007 The Authors Journal compilation ª 2007 FEBS Metabolism and Energetics Abstracts D3-23 The V-ATPase as drug target D3-25 Antifungal effect of pleurocidin and its M Huss1, T Bender2, S... Hajihassan, A Rabbani Chadegani and M Yousef Masboogh Institute of Biochemistry and Biophysics, Tehran, ISLAMIC REPUBLIC OF IRAN Mitoxantrone is a synthetic antineoplastic agent widely used in treatment of various cancers It binds to DNA by intercalation and inhibits DNA and RNA synthesis Also it is a potent inhibitor of topoisomerase II so leads to single and double strand DNA breaks In the present... increased and the highest effect is observed at 35 lM It is therefore suggested that the Mitoxantrone binds to soluble chromatin and induces chromatin aggregation ª 2007 The Authors Journal compilation ª 2007 FEBS Metabolism and Energetics D3-15 Binding of antitumor antibiotic, idarubicin, to histone H1 in soluble chromatin Z Zahraie, A Rabbani Chadegani and M Yousef Masboogh Institute of Biochemistry and. .. the matrix macromolecules and enhance invasion In order to examine whether gene expression of PGs and MMPs is related with breast cancer and invasive potential, we performed in vitro studies on a panel of epithelial breast cancer cell lines (ER-positive and -negative) as well functional invasion assays The obtained results clearly showed activated PDGFRb and that the PI3/Akt and ERK1/2 signalling pathways... Authors Journal compilation ª 2007 FEBS Metabolism and Energetics Abstracts D3-7 Blockade of HERG potassium channel by sibutramine HCl D3-9 Reversal of MDR by synthetic and natural compounds in drug resistant MCF-7 cell lines E Kim, S Park, K Kim and H Lee Korea Institute of Toxicology, Daejeon, REPUBLIC OF KOREA ¨ ¨ ¨ U Gunduz1, M Demirel Kars1, O Darcansoy _ ¸ eri1 and Is J Molnar2 1 Middle East Technical... pathogenic yeasts Benzoic and cinnamic acid derivatives were tested for biocidal activity against standard strains of Candida spp and Cryptococcus neoformans Minimum inhibitory concentrations (MIC 25, 50, 75 and 99) of the most potent compounds (>98% cell death at 5 mM) were determined Respective MIC values were determined for antifungal drugs Amphotericin B, Fluconazole, and Itraconazole Optimal concentrations... glomerulosclerosis and capillary basement membrane thickening suggest that mGS and H-GS can be used as a new marker of early and late diabetic nephropathy (FONDECYT 1051057; PBCT5; DID UACH SB2005-2) ª 2007 The Authors Journal compilation ª 2007 FEBS Metabolism and Energetics D5-9 In vivo determination of the flux control coefficients of the enzymes involved in glycogen synthesis A Preller, D Quiroga, C A M Wilson and. .. 79–85 235 Abstracts Metabolism and Energetics D3-19 New cellular and humural immune-targeted mechanisms by VIP/ADNP neuropeptides with prospective therapeutic uses D3-21 Glucosyl ceramide synthase in Alzheimer’s brain correlates with abnormal increase in long chain ceramides D Pozo1, F J Quintana2, E Gonzalez-Rey1, ´ R Fernandez-Montesinos1, J L Herrera1, A Chorny3, I Gozes4, I R Cohen5 and M Delgado6 1... support the hypothesis GCS insufficiency is a factor in AD and constitutes a new and important target for therapy D3-20 C-kit activity is dually involved in the mechanism of automatic rhythmic contractions of human myometrium C F Vidulescu, L Caravia, A Curici, L Ceafalan Suciu, A Popa and L M Popescu Carol Davila University of Medicine and Pharmacy, Bucharest, ROMANIA D3-22 Quercetin and other flavonoids . TB and Tews I PNAS. 103(51): 19284–19289 Metabolism and Energetics Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 221 D1-5 Heavy metal and. positive, bacil and spore-forming isolates (K1 and D3) were chosen, and the produc- tion of exracellular enzymes a-amylase and protease was studied and these