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ATP-driven efflux pumps such as phosphoglycoprotein-170 (P-gp), multidrug-resistance-associated protein-2 (MRP-2), or breast cancer resistance protein (BCRP) play a crucial role in limiting the efficacy of tumor pharmacotherapy. Selected flavonoids have been suggested to inhibit individual efflux-transporters and to act therefore as multidrug-resistance reversing agents. In the present study it is shown that the flavonoid chrysin acts as a potent inhibitor of P-gp, MRP-2, and BCRP in Caco-2 colon carcinoma cells. As a consequence, cells accumulated higher rates of the apoptosis-inducing chemotherapeutic topotecan in the presence of chrysin, even though under these conditions the expression of the transporters was markedly increased. Interestingly, in spite of the enhanced cellular drug accumulation the topotecan-induced apoptosis, assessed according to DNA-fragmentation, chromatin condensation, and by determination of sub-G1 peaks using fluorescence-assisted-cell sorting (FACS), was potently inhibited by chrysin. Suggested transport-independent apoptosis inhibiting activities of ATP-binding cassette (ABC)-transporters, such as the inhibition of caspases, were shown to be necessary for the inhibition of topotecan-induced apoptosis and were found to be associated with stabilization of beta-catenin especially in the cytosol. Inhibition of topotecan-induced intracellular acidification, however, was proven not to prevent caspase-activation and apoptosis. In conclusion, our studies show that chrysin in spite of raising the cellular concentrations of topotecan potently inhibits the apoptosis-inducing activities of the anti-tumor drug. Inhibition of caspase-activation was identified as the underlying mechanism and is suggested to be caused by transport-independent functions of ABC-transporters., (2010 Elsevier Inc. All rights reserved.)

The Birch reduction of aromatic rings to cyclohexadiene compounds is widely used in chemical synthesis and requires solvated electrons, the most potent reductants known in organic chemistry. Benzoyl-coenzyme A (CoA) reductases (BCR) are key enzymes in the anaerobic bacterial degradation of aromatic compounds and catalyze an analogous reaction under physiological conditions. Class I BCRs are FeS enzymes and couple the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA) to a stoichiometric ATP hydrolysis. Here, we report on a tungsten-containing class II BCR from Geobacter metallireducens that catalyzed the fully reversible, ATP-independent dearomatization of benzoyl-CoA to dienoyl-CoA. BCR additionally catalyzed the disproportionation of dienoyl-CoA to benzoyl-CoA/monoenoyl-CoA and the four- and six-electron reduction of benzoyl-CoA in the presence of a reduced low-potential bridged 2,2'-bipyridyl redox dye. Reversible redox titration experiments in the presence of this redox dye revealed a midpoint potential of E(0)' = -622 mV for the benzoyl-CoA/dienoyl-CoA couple, which is far below the values of other known reversible substrate/product redox couples in enzymology. This work demonstrates the efficiency of reversible metalloenzyme catalysis, which in chemical synthesis can only be achieved under essentially irreversible conditions.

Gaining knowledge in the field of health promotion may help physiotherapists acquire a new understanding of the way movement promotes health, as related to environmental influences. The aim of the present study was to describe how physiotherapists working in a programme of health promotion in compulsory school understand and approach their work. Seven physiotherapists in primary care, working partly with health promotion in compulsory school, were interviewed. A descriptive design of qualitative content analysis was chosen to analyse interviews that had already been conducted in a local evaluation of the programme. This resulted in a main theme, described as Perceiving and Supporting Coherence. This understanding and approach were discussed in relation to challenges of a historically embedded constitution of physiotherapy when entering the area of health promotion in compulsory school.

Glutaryl-coenzyme A dehydrogenases (GDHs) involved in amino acid degradation were thought to catalyze both the dehydrogenation and decarboxylation of glutaryl-coenzyme A to crotonyl-coenzyme A and CO(2). Recently, a structurally related but nondecarboxylating, glutaconyl-coenzyme A-forming GDH was characterized in the obligately anaerobic bacteria Desulfococcus multivorans (GDH(Des)) which conserves the free energy of decarboxylation by a Na(+)-pumping glutaconyl-coenzyme A decarboxylase. To understand the distinct catalytic behavior of the two GDH types on an atomic basis, we determined the crystal structure of GDH(Des) with and without glutaconyl-coenzyme A bound at 2.05 and 2.1 A resolution, respectively. The decarboxylating and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr --> Val exchange strictly conserved between the two GDH types. As a result, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved arginine is stronger in GDH(Des) (short and planar bidentate hydrogen bond) than in the decarboxylating human GDH (longer and monodentate hydrogen bond), which is corroborated by molecular dynamics studies. The identified structural changes prevent decarboxylation (i) by strengthening the C4-C5 bond of glutaconyl-coenzyme A, (ii) by reducing the leaving group potential of CO(2), and (iii) by increasing the distance between the C4 atom (negatively charged in the dienolate transition state) and the adjacent glutamic acid.

Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a sigma54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.

Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (K(m) = 24 +/- 4 microM). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an alpha(2)beta(2)-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The alphabeta unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S](0/+1) and three [4Fe-4S](+1/+2) clusters per alphabeta unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W- cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.

Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model organism Geobacter metallireducens identified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in the degradation of aromatic compounds in obligately anaerobic bacteria, differential membrane protein abundance levels from G. metallireducens grown on benzoate and acetate were determined by the MS-based spectral counting approach. A total of 931 proteins were identified by combining one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with liquid chromatography-tandem mass spectrometry. Several membrane-associated proteins involved in the degradation of aromatic compounds were newly identified including proteins with similarities to modules of NiFe/heme b-containing and energy-converting hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and a tungstate ATP-binding cassette transporter system. The transcriptional regulation of differentially expressed genes was analyzed by quantitative reverse transcription-PCR; in addition benzoate-induced in vitro activities of hydrogenase and nitrate reductase were determined. The results obtained provide novel insights into the poorly understood degradation of aromatic compounds in obligately anaerobic bacteria.

In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO(2). In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDH(Geo)) (Fe[III] reducing) and Desulfococcus multivorans (GDH(Des)) (sulfate reducing). GDH(Geo) was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDH(Des) was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDH(Geo) catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDH(Des) catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDH(Geo), the K(m) was 30 +/- 2 microM and the V(max) was 3.2 +/- 0.2 micromol min(-1) mg(-1), and for GDH(Des), the K(m) was 52 +/- 5 microM and the V(max) was 11 +/- 1 micromol min(-1) mg(-1). GDH(Des) but not GDH(Geo) was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDH(Geo) but are missing in GDH(Des). The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

Dearomatizing benzoyl-coenzyme A reductases (BCR) from facultatively anaerobic bacteria are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of benzoyl-CoA (BCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA). A Birch reduction mechanism involving alternate electron transfer and protonation steps has been proposed for BCR. In this work we reacted BCoA in H2O and D2O, and d5-BCoA in H2O with BCR and the second enzyme of the pathway, dienoyl-CoA hydratase (DCH). The 1,4 hydration product formed from the dienoyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, was analyzed by several NMR techniques. The results obtained indicate that BCR stereoselectively forms the trans-dienoyl-CoA product, and DCH stereoselectively catalyzes a trans-1,4 water addition. Moreover, unexpected proton exchanges at C-2 and C-6 were observed. They indicate that a free radical intermediate with an unusual low pKa is formed during BCR catalysis. This finding provides evidence for the proposed Birch reduction mechanism of BCR and is in agreement with the established radical mechanism of homologous alpha-hydroxyacyl-CoA dehydratases.

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 +/- 15 kDa and a unique alphaalpha'beta(2) subunit composition, with alpha and alpha' representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and beta representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr(394) of the alpha subunit. In contrast, the alpha' subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K(m) values (1.7 and 2.7 microM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K(ic) (competitive inhibition) value of 18 +/- 9 microM and a K(iu) (uncompetitive inhibition) value of 235 +/- 20 microM. A putative functional model for an unusual PCMH enzyme is presented.

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a member of the xanthine oxidase (XO) family of molybdenum cofactor containing enzymes and catalyzes the irreversible removal of a phenolic hydroxy group by reduction, yielding benzoyl-CoA and water. In this work the effects of various activity modulating compounds were characterized by kinetic, electron paramagnetic resonance (EPR) spectroscopic, and X-ray crystallographic studies. 4-HBCR was readily inactivated by cyanide and by the reducing agents titanium(III) citrate and dithionite; in contrast, reduced viologens had no inhibitory effect. Cyanide inhibition occurred in both the oxidized and reduced state of 4-HBCR. In the reduced state, cyanide-inhibited 4-HBCR was reactivated by simple oxidation. In contrast, reactivation from the oxidized state was only achieved in the presence of sulfide. Dithionite-inhibited 4-HBCR was reactivated by oxidation, whereas inhibition by titanium(III) citrate was irreversible. The previously reported inhibitory effect of azide could not be confirmed; instead, azide rather protected the enzyme from inactivation by titanium(III) citrate. The EPR spectra of the Mo(V) states were nearly identical in the noninhibited methyl viologen and in the dithionite-inhibited states of 4-HBCR; they exhibited a hyperfine splitting due to magnetic coupling with two solvent-exchangeable protons. The cyanide-treated enzyme showed the typical desulfo-inhibited Mo(V) EPR signal in D 2O, whereas in H 2O the hyperfine splitting was altered but indicated no loss of Mo(V)-proton interactions. The structures of dithionite- and azide-bound 4-HBCR were solved at 2.1 and 2.2 A, respectively. Both dithionite and azide bound directly to equatorial ligation sites of the Mo atom. The results obtained revealed further insights into the active site of an unusual member of the XO family of molybdenum cofactor containing enzymes.

In aerobic and facultatively anaerobic bacteria, the degradation of para-cresol (p-cresol) involves the initial hydroxylation to p-hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p-cresol methylhydroxylase (PCMH; alpha(2)beta(2) composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p-hydroxybenzoate, the formation of p-hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p-cresol by addition to fumarate, yielding p-hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p-cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The alpha subunit of the enzyme was present in two isoforms, suggesting an alphaalpha'beta(2) composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p-cresol oxidation) or to menaquinone (in the case of p-hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p-cresol metabolism, p-hydroxybenzoate-CoA ligase, and p-hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p-cresol metabolism (pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.

Background: Hydroxyethyl starches (HES) accumulate in the circulation when administered repeatedly. Accumulation is thought to be partly responsible for undesirable effects (tissue storage, blood coagulation impairment, and itching). HES 130/0.42 with low molecular weight and a low level of substitution has recently been developed in order to reduce those risks., Methods: In healthy volunteers, the pharmacokinetics of HES 130/0.42/6:1 were investigated using a crossover design with HES 200/0.5 serving as control. Fifty grams of either HES were administered in 4 h day-1 for a period of five consecutive days. HES serum concentrations were used for computation of pharmacokinetic coefficients. Change between the first and fifth infusion in the area under the concentration curve (AUC) served as the primary measurement., Results: Although the circulation was freed from the load with HES 130/0.42 within 20 h after end of the previous infusion, the amount of HES 200/0.5 increased continuously from one administration to the other. AUC and elimination half-life (t1/2) were significantly lower with HES 130/0.42. AUC and t1/2 of HES 200/0.5 showed an increase between the first and the fifth administration whereas only a minimal shift was present with HES 130/0.42. Haemodilution via HES 200/0.5 did not change over time., Conclusions: Repeated administration of HES 130/0.42 shows no accumulation and fewer tendencies to time-dependent changes in pharmacokinetic parameters than HES 200/0.5. The improved reproducibility may improve drug safety, particularly as the accumulation of residual starch with HES 200/0.5 does not contribute to the colloid's volume effect, but may rather increase the risk of undesired reactions.

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

The degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP-dependent ring reductase for the dearomatization of the activated key intermediate benzoyl-coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens. The gene coding for a putative carboxylic acid-CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate-CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP-dependent benzoyl-CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate-induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl-CoA, which is then reductively dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA, followed by beta-oxidation reactions to acetyl-CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.

Aerobic bacteria use molecular oxygen as a common co-substrate for key enzymes of aromatic metabolism. In contrast, in anaerobes all oxygen-dependent reactions are replaced by a set of alternative enzymatic processes. The anaerobic degradation of phenol to a non-aromatic product involves enzymatic processes that are uniquely found in the aromatic metabolism of anaerobic bacteria: (i) ATP-dependent phenol carboxylation to 4-hydroxybenzoate via a phenylphosphate intermediate (biological Kolbe-Schmitt carboxylation); (ii) reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA; and (iii) ATP-dependent reductive dearomatization of the key intermediate benzoyl-CoA in a 'Birch-like' reduction mechanism. This review summarizes the results of recent mechanistic studies of the enzymes involved in these three key reactions.

The molybdenum enzymes 4-hydroxybenzoyl-CoA reductase and pyrogallol-phloroglucinol transhydroxylase and the tungsten enzyme acetylene hydratase catalyze reductive dehydroxylation reactions, i.e., transhydroxylation between phenolic residues and the addition of water to a triple bond. Such activities are unusual for this class of enzymes, which carry either a mononuclear Mo or W center. Crystallization and subsequent structural analysis by high-resolution X-ray crystallography has helped to resolve the reaction centers of these enzymes to a degree that allows us to understand the interaction of the enzyme and the respective substrate(s) in detail, and to develop a concept for the respective reaction mechanism, at least in two cases.

Background: The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties., Methods: Thirty pigs were infused with three different HES solutions (20 ml kg(-1)) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model., Results: Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre(-1), P<0.001, 1701 (321) g min litre(-1), P<0.001] than HES 130 [1156 (223) g min litre(-1)] and alpha half life (t(alpha)(1/2)) was longer for HES 500 [53.8 (8.6) min, P<0.01] and HES 900 [57.1 (12.3) min, P<0.01] than for HES 130 [39.9 (10.7) min]. Beta half life (t(beta)(1/2)), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]., Conclusions: In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight low-substituted HES might result in a longer lasting volume effect.

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.

The high resonance energy of the benzene ring is responsible for the relative resistance of aromatic compounds to biodegradation. Nevertheless, bacteria from nearly all physiological groups have been isolated which utilize aromatic growth substrates as the sole source of cell carbon and energy. The enzymatic dearomatization of the benzene nucleus by microorganisms is accomplished in two different manners. In aerobic bacteria the aromatic ring is dearomatized by oxidation, catalyzed by oxygenases. In contrast, anaerobic bacteria attack the aromatic ring by reductive steps. Key intermediates in the anaerobic aromatic metabolism are benzoyl-CoA and compounds with at least two meta-positioned hydroxyl groups (resorcinol, phloroglucinol and hydroxyhydroquinone). In facultative anaerobes, the reductive dearomatization of the key intermediate benzoyl-CoA requires a stoichiometric coupling to ATP hydrolysis, whereas reduction of the other intermediates is readily achieved with suitable electron donors. Obligately anaerobic bacteria appear to use a totally different enzymology for the reductive dearomatization of benzoyl-CoA including selenocysteine- and molybdenum- containing enzymes., (Copyright 2005 S. Karger AG, Basel.)

The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis.

The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP). The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2 is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones, cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

Recently, the PAT family of proton-dependent amino acid transporters has been identified as a novel class of mammalian amino acid symporters. PAT1 and PAT2 members mediate electrogenic uptake of small, neutral amino acids and derivatives by cotransport of protons. Analysis of the structural requirements for substrate recognition by PAT1 identified that a free amino group in a substrate is not essential for recognition. We therefore hypothesized that PAT1 and its ortholog PAT2 may also be able to recognize and transport the homologous short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. We examined in Xenopus laevis oocytes whether the SCFAs interact with the transporter by employing flux studies, electrophysiology and intracellular pH recordings. SCFAs did not induce positive inward currents but inhibited glycine-induced transport currents. PAT-mediated uptake of radiolabeled proline was also dose-dependently reduced by SCFA and could be described by first order competition kinetics with apparent Ki-values for butyrate of 6.0 +/- 0.7 and 7.6 +/- 1.3 mM for PAT1 and PAT2, respectively. Acetate as well as propionate uptake was significantly enhanced in oocytes expressing PAT1 or PAT2. An electroneutral H+/SCFA symport mode was demonstrated by recording intracellular pH changes under voltage clamp conditions with rate constants for the initial intracellular acidification in the presence of SCFAs significantly increased in PAT-expressing oocytes. In conclusion, our data demonstrate that the PAT1 and PAT2 proteins are capable to transport selected SCFAs in an electroneutral and the homologous amino acids in an electrogenic mode and are therefore a paradigm for bifunctional solute carriers.

Background and Aims: Amino acid (and related drug) absorption across the human small intestinal wall is an essential intestinal function. Despite the revelation of a number of mammalian genomes, the molecular identity of the classic Na(+)-dependent imino acid transporter (identified functionally in the 1960s) remains elusive. The aims of this study were to determine whether the recently isolated complementary DNA hPAT1 (human proton-coupled amino acid transporter 1), or solute carrier SLC36A1, represents the imino acid carrier; the Na(+) -dependent imino acid transport function measured at the brush-border membrane of intact intestinal epithelia results from a close functional relationship between human proton-coupled amino acid transporter-1 and N(+) /H(+) exchanger 3 (NHE3)., Methods: PAT1 function was measured in isolation ( Xenopus laevis oocytes) and in intact epithelia (Caco-2 cell monolayers and rat small intestine) by measurement of amino acid and/or H(+) influx. Tissue and membrane expression of PAT1 were determined by reverse-transcription polymerase chain reaction and immunohistochemistry., Results: PAT1-specific immunofluorescence was localized exclusively to the luminal membrane of Caco-2 cells and human and rat small intestine. The substrate specificity of hPAT1 is identical to that of the imino acid carrier. In intact epithelia, PAT1-mediated amino acid influx is reduced under conditions in which NHE3 is inactive., Conclusions: The identification in intact epithelia of a cooperative functional relationship between PAT1 (H(+) /amino acid symport) and NHE3 (N(+) /H(+) exchange) explains the apparent Na + dependence of the imino acid carrier in studies with mammalian intestine. hPAT1 is the high-capacity imino acid carrier localized at the small intestinal luminal membrane that transports nutrients (imino/amino acids) and orally active neuromodulatory agents (used to treat affective disorders).

We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

The mammalian intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides from the gut lumen into intestinal epithelial cells and acts in parallel with amino acid transporters. Here we address the importance of the PEPT1 orthologue PEP-2 for the assimilation of dietary protein and for overall protein nutrition in Caenorhabditis elegans. pep-2 is expressed specifically along the apical membrane of the intestinal cells, and in pep-2 deletion mutant animals, uptake of intact peptides from the gut lumen is abolished. The consequences are a severely retarded development, reduced progeny and body size, and increased stress tolerance. We show here that pep-2 cross-talks with both the C. elegans target of rapamycin (TOR) and the DAF-2/insulin-signaling pathways. The pep-2 mutant enhances the developmental and longevity phenotypes of daf-2, resulting, among other effects, in a pronounced increase in adult life span. Moreover, all aspects of a weak let-363/TOR RNA interference phenotype are intensified by pep-2 deletion, indicating that pep-2 function upstream of TOR-mediated nutrient sensing. Our findings provide evidence for a predominant role of the intestinal peptide transporter for the delivery of bulk quantities of amino acids for growth and development, which consequently affects signaling pathways that regulate metabolism and aging.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.

The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy. In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism. An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate. Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted. The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii. When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements. The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium. In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements. In extracts of cells grown on benzoate in the presence of [75Se]selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate. These results suggest that the dearomatization process in D. multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.

Benzoyl-CoA reductase (BCR) from the denitrifying bacterium Thauera aromatica catalyzes the ATP driven two-electron reduction of the aromatic moiety of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene (2 ATP/2 e(-)). The enzyme contains two similar but nonidentical ATP-binding sites of the acetate kinase/sugar kinase/Hsp70/actin family. To obtain further insights into the overall catalytic cycle of BCR, the binding affinities and stoichiometries of all substrates as well as their effects on reduction kinetics were studied by stopped-flow UV/vis spectroscopy, freeze-quench EPR spectroscopy, and equilibrium dialysis. BCR bound maximally two nucleotides and a single BCoA. The binding of a single nucleotide induced a molecular switch (BCR --> BCR) as evidenced as follows: (i) the reduction rate of BCR by sulfoxide radical anion was significantly decreased in the nucleotide-bound state, (ii) the binding of BCoA to the reduced enzyme strictly depended on bound nucleotides, and (iii) the nucleotide binding affinities increased up to 60-fold compared to the steady-state values. The "ATP-binding switch" is distinguished from the previously described "low-spin/high-spin switch" of a [4Fe-4S] cluster which strictly depends on ATP hydrolysis. The two nucleotide binding sites were occupied sequentially; binding constants of the two sites differed by a factor of 10-40. The kinetic data obtained suggest that the ATP-binding switch is a rather fast process (>100 s(-)(1)) with a switch equilibrium constant of 54 +/- 10. In contrast, the reverse switch back of the MgADP-bound enzyme (BCR --> BCR) is considered rate-limiting in the overall catalytic cycle of BCR (4 +/- 1 s(-)(1)). The binding of nucleotides did not affect the redox potential of the [4Fe-4S](+1/+2) clusters; the switch is rather considered to alter the kinetics of internal electron transfer. Implications for the overall catalytic cycle of benzoyl-CoA reductase are discussed and compared with other ATP-hydrolyzing enzymes.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a central enzyme in the metabolism of phenolic compounds in anaerobic bacteria. The enzyme catalyzes the reductive removal of the phenolic hydroxyl group from 4-hydroxybenzoyl-CoA, yielding benzoyl-CoA and water. 4-HBCR belongs to the xanthine oxidase (XO) family of molybdenum enzymes which occur as heterodimers, (alphabetagamma)(2). 4-HBCR contains two molybdopterins, four [2Fe-2S] and two [4Fe-4S] clusters and two FADs. A low-potential Allochromatium vinosum-type ferredoxin containing two [4Fe-4S] clusters serves as an in vivo electron donor for 4-HBCR. In this work, the oxygen-sensitive proteins 4-HBCR and the ferredoxin (TaFd) from Thauera aromatica were crystallized under anaerobic conditions. 4-HBCR crystallized with PEG 4000 and MPD as precipitant diffracted to about 1.6 A resolution and the crystals were highly suitable for X-ray structure analysis. Crystals of TaFd were obtained with (NH(4))(3)PO(4) as precipitant and revealed a solvent content of 77%, which is remarkably high for a small soluble protein. The structure of TaFd was solved at 2.9 A resolution by the molecular-replacement method using the highly related structure of the ferredoxin (CvFd) from A. vinosum as a model. Structural changes between the two ferredoxins around the [4Fe-4S] cluster can be correlated with their different redox potentials.

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

We report the molecular and functional characterisation of a novel peptide transporter from zebrafish, orthologue to mammalian and avian PEPT1. Zebrafish PEPT1 is a low-affinity/high-capacity system. However, in contrast to higher vertebrate counterparts in which maximal transport activity is independent of extracellular pH, zebrafish PEPT1 maximal transport rates unexpectedly increase at alkaline extracellular pH. Zebrafish pept1 is highly expressed in the proximal intestine since day 4 post-fertilisation, thus preceding functional maturation of the gut, first feeding and complete yolk resorption. Zebrafish PEPT1 might help to understand the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, zebrafish pept1 can be a useful marker for screening mutations that affect gut regionalisation, differentiation and morphogenesis.

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.

We recently cloned and functionally characterized two novel proton/amino acid transporters (PAT1 and PAT2) from mouse. Here we report the isolation of the corresponding cDNAs of the human orthologues and one additional mouse and human PAT-like transporter cDNA, designated PAT3. The PAT proteins comprise 470 to 483 amino acids. The mouse PAT3 mRNA is expressed in testis of adult mice. In the human and mouse genomes the genes of the PAT transporters (designated SLC36A1-3 and Slc36a1-3, respectively) are clustered on human chromosome 5q33.1 and in the syntenic region of mouse chromosome 11B1.3. PAT-like transporter genes are present as well in the genomes of other eukaryotic organisms such as Drosophila melanogaster and Caenorhabditis elegans. For the PAT3 subtype transporter, we could not yet identify its function. The human PAT1 and PAT2 transporters when functionally expressed in Xenopus laevis oocytes show characteristics similar to those of their mouse counterparts.

The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.

The use of many halogenated alkanes such as carbon tetrachloride (CCl4), chloroform (CHCl3) or iodoform (CHI3), has been banned or severely restricted because of their distinct toxicity. Yet CCl4 continues to provide an important service today as a model substance to elucidate the mechanisms of action of hepatotoxic effects such as fatty degeneration, fibrosis, hepatocellular death, and carcinogenicity. In a matter of dose,exposure time, presence of potentiating agents, or age of the affected organism, regeneration can take place and lead to full recovery from liver damage. CCl4 is activated by cytochrome (CYP)2E1, CYP2B1 or CYP2B2, and possibly CYP3A, to form the trichloromethyl radical, CCl3*. This radical can bind to cellular molecules (nucleic acid, protein, lipid), impairing crucial cellular processes such as lipid metabolism, with the potential outcome of fatty degeneration (steatosis). Adduct formation between CCl3* and DNA is thought to function as initiator of hepatic cancer. This radical can also react with oxygen to form the trichloromethylperoxy radical CCl3OO*, a highly reactive species. CCl3OO* initiates the chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids, in particular those associated with phospholipids. This affects the permeabilities of mitochondrial, endoplasmic reticulum, and plasma membranes, resulting in the loss of cellular calcium sequestration and homeostasis, which can contribute heavily to subsequent cell damage. Among the degradation products of fatty acids are reactive aldehydes, especially 4-hydroxynonenal, which bind easily to functional groups of proteins and inhibit important enzyme activities. CCl4 intoxication also leads to hypomethylation of cellular components; in the case of RNA the outcome is thought to be inhibition of protein synthesis, in the case of phospholipids it plays a role in the inhibition of lipoprotein secretion. None of these processes per se is considered the ultimate cause of CCl4-induced cell death; it is by cooperation that they achieve a fatal outcome, provided the toxicant acts in a high single dose, or over longer periods of time at low doses. At the molecular level CCl4 activates tumor necrosis factor (TNF)alpha, nitric oxide (NO), and transforming growth factors (TGF)-alpha and -beta in the cell, processes that appear to direct the cell primarily toward (self-)destruction or fibrosis. TNFalpha pushes toward apoptosis, whereas the TGFs appear to direct toward fibrosis. Interleukin (IL)-6, although induced by TNFalpha, has a clearly antiapoptotic effect, and IL-10 also counteracts TNFalpha action. Thus, both interleukins have the potential to initiate recovery of the CCl4-damaged hepatocyte. Several of the above-mentioned toxication processes can be specifically interrupted with the use of antioxidants and mitogens, respectively, by restoring cellular methylation, or by preserving calcium sequestration. Chemicals that induce cytochromes that metabolize CCl4, or delay tissue regeneration when co-administered with CCl4 will potentiate its toxicity thoroughly, while appropriate CYP450 inhibitors will alleviate much of the toxicity. Oxygen partial pressure can also direct the course of CCl4 hepatotoxicity. Pressures between 5 and 35 mmHg favor lipid peroxidation, whereas absence of oxygen, as well as a partial pressure above 100 mmHg, both prevent lipid peroxidation entirely. Consequently, the location of CCl4-induced damage mirrors the oxygen gradient across the liver lobule. Mixed halogenated methanes and ethanes, found as so-called disinfection byproducts at low concentration in drinking water, elicit symptoms of toxicity very similar to carbon tetrachloride, including carcinogenicity.

Aromatic compounds and hydrocarbons have in common a great stability due to resonance energy and inertness of CbondH and CbondC bonds. It has been taken for granted that the metabolism of these compounds obligatorily depends on molecular oxygen. Oxygen is required first to introduce hydroxyl groups into the substrate and then to cleave the aromatic ring. However, newly discovered bacterial enzymes and reactions involved in oxidation of aromatic and hydrocarbon compounds to CO(2) in the complete absence of molecular oxygen have been discovered. Of special interest are two reactions: the reduction of the aromatic ring of benzoyl-coenzyme A and the addition of fumarate to hydrocarbons. These reactions transform aromatic rings and hydrocarbons into products that can be oxidized via more conventional beta-oxidation pathways.

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant (Kt) value of 1.1 mM. Several dipeptides, cefadroxil, and delta-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 +/- 29 omega x cm(2). The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.

Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-). Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [(14)C]ADP to [(14)C]ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with [gamma-(32)P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.