Binding region of alanopine dehydrogenase predicted by unbiased molecular dynamics simulations of ligand diffusion.

Opine dehydrogenases catalyze the reductive condensation of pyruvate with L-amino acids. Biochemical characterization of alanopine dehydrogenase from Arenicola marina revealed that this enzyme is highly specific for L-alanine. Unbiased molecular dynamics simulations with a homology model of alanopine dehydrogenase captured the binding of L-alanine diffusing from solvent to a putative binding region near a distinct helix-kink-helix motif. These results and sequence comparisons reveal how mutations and insertions within this motif dictate the L-amino acid specificity.

Control of D-octopine formation in scallop adductor muscle as revealed through thermodynamic studies of octopine dehydrogenase.

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus (Linné, 1758), catalyses the NADH-dependent condensation of l-arginine and pyruvate to d-octopine, NAD(+) and water during escape swimming and subsequent recovery. During exercise, ATP is mainly provided by the transphosphorylation of phospho-l-arginine and to some extent by anaerobic glycolysis. NADH resulting from the glycolytic oxidation of 3-phosphoglyceraldehyde to 1,3-bisphosphoglycerate is reoxidized during d-octopine formation. In some scallops d-octopine starts to accumulate during prolonged, strong muscular work, whereas in other species d-octopine formation commences towards the end of swimming and continues to rise during subsequent recovery. The activity of OcDH is regulated by a mandatory, consecutive mode of substrate binding in the order NADH, l-arginine and pyruvate, as demonstrated by isothermal titration calorimetry. The first regulatory step in the forward reaction comprises the binding of NADH to OcDH with a dissociation constant K(d) of 0.014±0.006 mmol l(-1), which reflects a high affinity and tight association of the apoenzyme with the co-substrate. In the reverse direction, NAD(+) binds first with a K(d) of 0.20±0.004 mmol l(-1) followed by d-octopine. The binary OcDH-NADH complex associates with l-arginine with a K(d) of 5.5±0.05 mmol l(-1). Only this ternary complex combines with pyruvate, with an estimated K(d) of approximately 0.8 mmol l(-1) as deduced from pyruvate concentrations determined in the muscle of exhausted scallops. At tissue concentrations of pyruvate between 0.5 and 1.2 mmol l(-1) in the valve adductor muscle of fatigued P. maximus, binding of pyruvate to OcDH plays the most decisive role in initiating OcDH activity and, therefore, in controlling the onset of d-octopine formation.

Insights into the mechanism of ligand binding to octopine dehydrogenase from Pecten maximus by NMR and crystallography.

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD(+), and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.

Thermodynamics of effector binding to hemocyanin: influence of temperature.

Hemocyanins are allosterically regulated oxygen carriers freely dissolved in the blood of many crustaceans. The natural modulator urate accumulates under hypoxic conditions in the hemolymph of Homarus vulgaris, and increases the oxygen affinity of the respiratory pigment. Other non-physiological effectors such as caffeine, dimethylxanthines and methylxanthines are also known to modulate the oxygen-binding properties of hemocyanin. In order to gain insight into the thermodynamic driving forces of these interactions we analyzed the binding of several urate analogs to dodecameric hemocyanin at different temperatures by employing isothermal titration calorimetry (ITC). All investigated non-physiological effectors including caffeine, dimethylxanthines and methylxanthines bind exothermically to hemocyanin and the binding processes are enthalpy driven. Furthermore, we demonstrated a strong temperature dependent binding of caffeine and dimethylxanthines to the hemocyanin of the European lobster and could show that the binding properties of the effectors to the urate-binding site depend on the hydrophobicity of a given molecule.

Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase.

Over the last decade, protein purification has become more efficient and standardized through the introduction of affinity tags. The choice and position of the tag, however, can directly influence the process of protein crystallization. Octopine dehydrogenase (OcDH) without a His tag and tagged protein constructs such as OcDH-His(5) and OcDH-LEHis(6) have been investigated for their crystallizability. Only OcDH-His(5) yielded crystals; however, they were multiple. To improve crystal quality, the cofactor NADH was added, resulting in single crystals that were suitable for structure determination. As shown by the structure, the His(5) tag protrudes into the cleft between the NADH and L-arginine-binding domains and is mainly fixed in place by water molecules. The protein is thereby stabilized to such an extent that the formation of crystal contacts can proceed. Together with NADH, the His(5) tag obviously locks the enzyme into a specific conformation which induces crystal growth.

Redox regulation of mitochondrial sulfide oxidation in the lugworm, Arenicola marina.

Sulfide oxidation in the lugworm, Arenicola marina (L.), is most likely localized in the mitochondria, which can either produce ATP with sulfide as a substrate or detoxify it via an alternative oxidase. The present study identified selective activators of the energy-conserving and the detoxifying sulfide oxidation pathways respectively. In the presence of the ROS scavengers glutathione (GSH) and ascorbate, isolated lugworm mitochondria rapidly oxidized up to 100 micromoll(-1) sulfide with maximal oxygen consumption rates but did not produce any ATP in the process. Under these conditions, salicylhydroxamic acid (SHAM), which is an inhibitor of the alternative oxidase of plant mitochondria, completely blocked oxygen consumption whereas inhibitors of complex III and IV had hardly any effect. By contrast, dehydroascorbate (DHA) enabled the mitochondria to gain ATP from sulfide oxidation even if the sulfide concentration far exceeded the threshold for inhibition of cytochrome oxidase. In the presence of dehydroascorbate, respiratory rates were independent of sulfide concentrations, with a respiratory control ratio of 2.1+/-0.2, and both oxygen consumption and ATP production were completely inhibited by myxothiazol and sodium azide but only marginally by SHAM. The present data indicate that a redox mechanism may contribute to the regulation of sulfide oxidation in lugworm mitochondria in vivo. Thus, mitochondria are presumably much more sulfide resistant in a cellular context than previously thought.

A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus.

Octopine dehydrogenase [N(2)-(D-1-carboxyethyl)-L-arginine:NAD(+) oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD(+), thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a "molecular ruler" mechanism.

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria.

Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body-wall tissue of the lugworm, Arenicola marina. A membrane-bound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide : quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese.

Modulatory effects of adenosine and adenine nucleotides on different heart preparations of the American lobster, Homarus americanus.

As shown previously, adenosine and the adenine nucleotides cause a rapid increase in heart rate (f(H)) and haemolymph velocity (v(HL)) when infused into intact American lobster (Homarus americanus). Here we compare the effects of adenosine and adenine nucleotides on different heart preparations in order to gain insight into their sites of action. In the semi-isolated (in situ) heart preparation where the heart is uncoupled from neural and hormonal influence AMP, ADP and ATP, but not adenosine increased contractile force. None of the purines altered f(H). Thus, the adenine nucleotides directly affect the myocardium and not the f(H)-setting cardiac ganglion. In cardioregulatory-denervated animals in which the cardiac ganglion only was severed from the central nervous system (CNS), purines caused a small and gradual increase in f(H), indicating that in vivo an alteration of f(H) arises indirectly through the central nervous system which in turn sends the information to the heart via the dorsal nerves. The gradual increase in f(H) of cardioregulatory-denervated animals may also result from neurohormones released into the circulatory system, although no significant changes in haemolymph concentration of dopamine, serotonin and octopamine were found during adenosine infusion. In semi-isolated (in situ) hearts adenine nucleotides also increased haemolymph flow, as a consequence of increased heart contractile force, but again adenosine had no effect. These data show that in vivo adenosine does not influence the myocardium, only the adenine nucleotides affect the myocardium directly. Obviously adenosine possesses an indirect effect, perhaps on cardio-arterial valves and arterial resistance, but other, as yet unidentified, modifying factors are also possible.

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L).

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.

Allosteric models for multimeric proteins: oxygen-linked effector binding in hemocyanin.

In many crustaceans, changing concentrations of several low molecular weight compounds modulates hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the pigment. The nonphysiological effector caffeine and the physiological modulator urate, the latter accumulating in the hemolymph of the lobster Homarus vulgaris during hypoxia, increase hemocyanin oxygen affinity and decrease cooperativity of oxygen binding. To derive a model that describes the mechanism of allosteric interaction between hemocyanin and oxygen in the presence of urate or caffeine, studies of oxygen, urate, and caffeine binding to hemocyanin were performed. Exposure of lobster hemocyanin to various pH values between 7.25 and 8.15 resulted in a decrease of p50. In this pH interval, p50 decreases from 95 to 11 Torr without effectors and from 49 to 6 Torr and from 34 to 5 Torr in the presence of 1 mM urate or caffeine, respectively. Thus, the allosteric effects induced by protons and urate or caffeine are coupled. In contrast, isothermal titration calorimetry did not reveal any differences in binding enthalpy (DeltaH degrees ) for urate or caffeine under either normoxic or hypoxic conditions at different pH values. Despite these apparently conflicting results, they can be explained by the nested MWC model if two different types of modulator binding sites are assumed, an allosteric and a nonallosteric type of site. Simulations of in vivo conditions with this model indicate that the naturally occurring modulator urate is physiologically relevant in H. vulgaris only during hypoxic conditions, i.e., either during environmental oxygen limitation or extensive exercise.

Extracellular and intracellular acid-base status with regard to the energy metabolism in the oyster Crassostrea gigas during exposure to air.

The acid-base status of extra- and intracellular fluids was studied in relation to the anaerobic energy metabolism in the adductor muscle, mantle, gills, and heart of the marine bivalve Crassostrea gigas after exposure to air for periods of 2, 4, 8, 12, 24, and 48 h. Such exposure was found to cause a significant reduction in the pH in the hemolymph (pH(e)) within the first 4 h. The decrease in the pHe was accompanied by elevated Pco2 values, causing [HCO3-] to rise (respiratory acidosis). Thereafter, the pHe fell at a lower rate, and this fall was partially compensated for by a further increase in [HCO3-] in the hemolymph. The increase in the [Ca] levels in the hemolymph indicates a mobilization of Ca2+ from CaCO3 and the involvement of bicarbonates in the buffering of pHe. The main anaerobic end-products that accumulated in the tissues during the first stages of anaerobiosis were alanine and succinate, at a ratio of about 2 : 1. Later on, propionate and acetate were also accumulated at significant rates. In contrast to the adductor muscle, gills, and mantle, opine production in the heart was significant after 12-24 h of exposure to air. Determination of intracellular pH (pHi) revealed that there is a close relationship between the rate of anaerobic end-product accumulation and the extent of intracellular acidosis in the adductor muscle, mantle, and gills. On the contrary, accumulation of anaerobic end-products in the heart did not cause any significant change in its pHi. The intracellular nonbicarbonate, nonphosphate buffering value (beta (NB,NPi)) was determined to be higher in the heart than in the other three tissues and thus probably plays a crucial role in stabilizing heart pHi during exposure to air.

Complementary DNA cloning and molecular evolution of opine dehydrogenases in some marine invertebrates.

The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

Energy metabolism and valve closure behaviour in the Asian clam Corbicula fluminea.

Since its invasion of Europe in the early 1980s, the Asian clam Corbicula fluminea has become very abundant in nearly all western river systems. Today this species is one of the most important biomass producers in the River Rhine. Monitoring the valve movements of C. fluminea over a period of 2 years revealed a circadian rhythm in summer, with extended periods (10-12 h) of valve closure, predominantly in the morning hours. Altogether valve movements were very scarce, frequently fewer than four movements per individual per day. Simultaneous measurements of heat dissipation and oxygen consumption (calorespirometry) revealed an intermittent metabolism in the clam. With the onset of valve closure, C. fluminea reduced its metabolic rate to 10% of the standard metabolic rate (SMR) measured when the valves were open. Nevertheless, this depressed metabolism remained aerobic for several hours, enabling the clam to save energy and substrates compared to the requirements of the tenfold higher SMR. Only during long-lasting periods of valve closure (more than 5-10 h) did the clams become anaerobic and accumulate succinate within their tissues (2 micromol g(-1) fresh mass). Succinate is transported into the mantle cavity fluid, where it reaches concentrations of 4-6 mmol l(-1). Because this succinate-enriched fluid must pass the gills when the valves open again, we suggest that this anaerobic end product is at least partly reabsorbed, thus reducing the loss of valuable substrates during anaerobiosis. Propionate was also produced, but only during experimental N2-incubation, under near-anoxic conditions. The intermittent metabolism of C. fluminea is discussed as an adaption to efficiently exploit the rare food supply, saving substrates by the pronounced metabolic depression during valve closure.

Targeting of endothelin receptors for molecular imaging of atherosclerosis in rabbits.

UNLABELLED: We wanted to determine whether a previously described in vivo accumulation of a (99m)Tc-labeled endothelin derivative in atherosclerotic lesions is mediated by binding to endothelin receptors. Furthermore, the expression of endothelin receptors in atherosclerotic lesions of 2 different rabbit animal models for atherosclerosis was to be evaluated to determine whether endothelin receptors generally are a suitable target for atherosclerosis imaging. METHODS: Normal vessels from untreated New Zealand White rabbits (NZW), balloon-denuded aortas from cholesterol-fed NZW, and atherosclerotic aortas from Watanabe Heritable Hyperlipidemic rabbits (WHHL) were used either as cross sections (cryosections) for receptor binding studies or for superfusion with a medium containing (125)I-labeled endothelin-1 or the (99m)Tc-labeled endothelin derivative. RESULTS: Cross sections of aortas from untreated NZW contained 45 +/- 11.10(6) endothelin A receptors per square millimeter and 55 +/-11.10(6).endothelin B receptors per square millimeter, cross sections of balloon-denuded aortas from cholesterol-fed NZW contained 106 +/- 16.10(6) endothelin A receptors per square millimeter and 27 +/- 16.10(6) endothelin B receptors per square millimeter, and cross sections of atherosclerotic aortas from WHHL contained 40 +/- 13.10(6) endothelin A receptors per square millimeter and 5 +/- 13.10(6) endothelin B receptors per square millimeter. Balloon-denuded aortas from cholesterol-fed NZW (366 +/- 132 amol.mm(-2), P < 0.001) and atherosclerotic aortas from WHHL (338 +/- 175 amol.mm(-2), P < 0.002) accumulated significantly more of the (99m)Tc-labeled endothelin derivative than did vessels from control animals (137 +/- 26 amol.mm(-2)). On the contrary, (125)I-labeled endothelin-1--bound receptor mediated to superfused aortas from untreated NZW (12 +/- 9 amol.mm(-2)) and to balloon-denuded aortas from cholesterol-fed NZW (19 +/- 5 amol.mm(-2)) but not to aortas from WHHL. This lack of receptor-specific accumulation of (125)I-endothelin-1 in atherosclerotic areas of WHHL aortas, and this receptor-specific accumulation in atherosclerotic balloon-denuded NZW aortas that does not significantly increase in comparison with normal aortas of untreated NZW, cause failure of endothelins to detect atherosclerotic lesions. CONCLUSION: Although the density and the ratio of endothelin receptor subtypes change because of the development of atherosclerotic lesions in rabbit aortas, endothelin receptor targeting for imaging of atherosclerosis is not suitable.