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The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.sup.1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family.sup.2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation.sup.3-5. Because SUCNR1 senses succinate as an immunological danger signal.sup.6--which has relevance for diseases including ulcerative colitis, liver fibrosis.sup.7, diabetes and rheumatoid arthritis.sup.3,8--it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo. High-resolution crystal structures of the rat succinate receptor SUCNR1 in an inactive confirmation, and the humanized rat SUCNR1 bound to an antagonist, provide insights into the structure of these receptors and the species selectivity of antagonist binding., Author(s): Matthias Haffke [sup.1] [sup.2] , Dominique Fehlmann [sup.3] , Gabriele Rummel [sup.1] , Jacques Boivineau [sup.1] , Myriam Duckely [sup.1] , Nina Gommermann [sup.2] , Simona Cotesta [sup.2] , [...]

NMR isotopic filiation of [sup.13]C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in Saccharomyces cerevisiae during anaerobic glucose fermentation. The assimilation of [[3-.sup.13]]C]aspartate led to the formation of [2,[3-.sup.13]C]malate and [2,[3-.sup.13]C]succinate, with equal levels of [sup.13]C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [[3-.sup.13]C]glutamate. The non-random distribution of [sup.13]C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed [sup.13]C distribution suggests that the succinate dehydrogenase (SDH) complex is not active during glucose fermentation. This hypothesis was tested by deleting the SDH1 gene encoding the flavoprotein subunit of the SDH complex. The growth, fermentation rate and metabolite profile of the sdh1 mutant were similar to those of the parental strain, demonstrating that SDH was indeed not active. Filiation experiments indicated the reductive branch of the TCA pathway was the main pathway for succinate production if aspartate was used as the nitrogen source, and that a surplus of succinate was produced by oxidative decarboxylation of 2-oxoglutarate if glutamate was the sole nitrogen source. Consistent with this finding, a kgd1 mutant displayed lower levels of succinate production on glutamate than on other nitrogen sources, and higher levels of oxoglutarate dehydrogenase activity were observed on glutamate. Thus, the reductive branch generating succinate via fumarate reductase operates independently of the nitrogen source. This pathway is the main source of succinate during fermentation, unless glutamate is the sole nitrogen source, in which case the oxidative decarboxylation of 2-oxoglutarate generates additional succinate.

The Gram-positive bacterium Bacillus subtilis has a complete set of enzymes for the tricarboxylic acid (TCA) cycle and can grow aerobically using most of the TCA cycle intermediates (malate, fumarate, succinate and citrate) as a sole carbon source. The B. subtilis genome sequence contains three paralogous two-component regulatory systems, CitST, DctSR and YufLM. CitST and DctSR activate the expression of a transporter of the [Mg.sup.2+]--citrate complex (CitM) and a fumarate and succinate transporter (DctP), respectively. These findings prompted an investigation of whether the YufL sensor and its cognate regulator, YufM, play a role in malate uptake. This paper reports that the YufM regulator shows in vitro binding to the promoter region of two malate transporter genes, maeN and yflS, and is responsible for inducing their expression in vivo. It was also found that inactivation of the yufM or maeN genes resulted in bacteria that could not grow in a minimal salts medium containing malate as a sole carbon source, indicating that the induction of the MaeN transporter by the YufM regulator is essential for the utilization of malate as a carbon source. Inactivation of the yufL gene resulted in the constitutive expression of MaeN. This expression was suppressed by reintroduction of the kinase domain of YufL, indicating that the YufL sensor is required for proper signal detection and signalling specificity. The authors propose that a phosphatase activity of YufL plays an important role in the YufLM two-component regulatory system. The studies reported here have revealed that members of a set of paralogous two-component regulatory systems in B. subtilis, CitST, DctSR and YufLM, are involved in a related function--uptake (and metabolism) of the TCA cycle intermediates--but with distinct substrate specificities.

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit, attached via an ester bond to the 3-OH group of the deoxysugar moiety. To investigate candidate genes responsible for the formation of this ester bond, a gene inactivation experiment was carried out in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976. An in-frame deletion was created in the coding sequence of the gene cloN2. The production of secondary metabolites in the wild-type and in the cloN2 mutant was analysed. The wild-type showed clorobiocin as the main product, whereas the cloN2 mutant accumulated a new aminocoumarin derivative, novclobiocin 104, lacking the pyrrole moiety at the 3-OH of the deoxysugar. In addition, free pyrrole-2-carboxylic acid accumulated in the culture extract of the cloN2 mutant. The structures of the metabolites were confirmed by NMR and LC-MS analysis. Clorobiocin production was successfully restored in the cloN2 mutant by introducing a replicative plasmid containing the cloN2 sequence. These results prove an involvement of cloN2 in the formation of the ester bond between the pyrrole moiety and the deoxysugar in clorobiocin biosynthesis. Furthermore, they indicate that the C-methylation at position 5 of the pyrrole moiety occurs after the attachment of pyrrole-2-carboxylic acid unit to the deoxysugar moiety.

Human paraoxonase (PON), a serum enzyme synthesized by the liver, catalyzes the hydrolysis of organophosphates (OPs) such as the toxic exon forms of the pesticide parathion (among which, paraoxon), of […]

The carboxylese is an integral membrane glycoprotein that uses vitamin K to modify clusters of glutamyl residues (glu's) to [gamma]-carboxylated glutamyl residues (gla's) post-translationally in vitamin K-dependent (VKD) proteins as they pass through the endoplesmic reticulum. Carboxylation is required for VKD protein functions in hemostasis, bone metabolism, growth control and signal transduction. Carboxylation of multiple glu residues is accomplished via a processive mechanism, which occurs with at least some order and involves carboxylation of the carboxylase. The carboxylase has a high affinity binding site for VKD proteins, which in most cases is a VKD propeptide sequence; it also appears to have a low affinity site for those glu's undergoing catalysis. The propeptide activates binding of the glu's; together, the two contact points between the carboxylese and VKD protein increase the affinity of the carboxylase for vitamin K. Biochemical mapping to identify where these events occur in the carboxylese remains a challenge, despite the availability of recombinant protein. The affinity of the carboxylase for the propeptide of several VKD proteins that are coexprsesed in liver varies over a 100-fold range. Treatment with anticoagulants such as warfarin that indirectly block carboxylation likely decreases the rate of VKD protein catalysis and increases the accumulation of VKD precursors, leading to a competitive state among these proteins, which results in the premature dissociation of undercarboxylated, inactive protein. J. Nutr. 130: 1877-1880, 2000. KEY WORDS: * vitamin K * vitamin K-dependent carboxylase

Our aim was to quantify the intestinal metabolic fate of dietary [alpha]-ketoglutarate (AKG). Female pigs (n = 6; 21 d old) were implanted with arterial, venous, portal and gastric catheters and an ultrasonic portal flow probe and fed a corn and soybean meal-based diet. On the day of the experiment, the pigs received a 4-h intragastric infusion of sodium AKG at a rate equivalent to 0, 2.5, 5 or 10% of dietary intake. The net portal AKG balance of the control and 2.5% treatments did not differ and were not different from zero. However, the net portal AKG balance of both the 5 [163/[micro]mol/(kg * h)] and 10% [159/[micro]mol/(kg * h)] treatments were greater (P < 0.05) than the control. Despite significant net AKG absorption at the 5 and 10% levels, the net portal appearance represented only 10.8 and 6.7%, respectively, of the enteral input. The net portal appearances of glutamate, glutamine, ammonia and the branched-chain amino acids were not affected by dietary AKG level. We conclude that the absorption of dietary AKG is limited in young pigs and does not change the net portal balance of amino acids or ammonia. KEY WORDS: * glutamate * ammonia * glutamate dehydrogenase * dicarboxylic acid transport * pigs

Research has been conducted on Saccharomyces cerevisiae lysine 213 and histidine 233. The role of these residues in Mn (super)2+ binding and in catalysis has been investigated and the results demonstrate that mutation of lysine 213 and histidine 233 affects catalysis and that they are required for proper Mn (super)2+ binding.

Research has been conducted on the isoxazolinopyrrole 2-carboxylates. The details of the solid-phase synthesis of these compounds which employs a traceless sulfone linker strategy are presented.

The characteristics of L-lactic acid transport across the trophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake of L-[[sup.14]C]lactic acid into basal membrane vesicles was [Na.sup.+] independent, and an uphill transport was observed in the presence of a pH gradient ([[[H.sup.+]].sub.out] > [[[H.sup.+]].sub.in]). L-[[sup.14]C]lactic acid uptake exhibited saturation kinetics with a [K.sub.m] value of 5.89 [+ or -] 0.68 mM in the presence of a pH gradient, p-Chloromercuribenzenesulfonate and [alpha]-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not. Mono- and dicarboxylic acids suppressed the initial uptake. In conclusion, L-lactic acid transport in the basal membrane is [H.sup.+] dependent and [Na.sup.+] independent, as is also the case for the brush-border membrane transport, and its characteristics resemble those of monocarboxylic acid transporters. However, there were several differences in the effects of inhibitors between basal and brush-border membrane vesicles, suggesting that the transporter(s) involved in L-lactic acid transport in the basal membrane of placental trophoblast may differ from those in the brush-border membrane. human placenta; trophoblast; monocarboxylic acid

Research has been conducted on 5-carboxyvanillate decarboxylase. The degradation pathway of this compound and the decarboxylase gene responsible for the compound transformation have been investigated and the results indicate that 5-carboxyvanillate decarboxylase is transformed to vanillate by Sphingomonas paucimobilis SYK-6.

Research has been conducted on the aldol products with allyl-protected hydroxy group. The conversion of these products into amino alcohols carried out via Curtius rearrangement is described.

Research has been conducted on the cooperative pinwheel chemosensor. The recognition properties of this chemosensor for dicarboxylates have been investigated and are reported.

Research has been conducted on the chiral alpha,beta,psi,omega-unsaturated bisphosphine oxide. Results indicate that after the treatment with lithium diisopropylamide and benzaldehyde this compound underwent Michael cyclization-aldol tandem reaction in producing endo-alpha,beta-unsaturated bisphosphine oxides.

Research has been conducted on the L-glutamic acid analogues. The synthesis and stereochemical characterization of 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acid isomers have been carried out and the details are reported.

Research has been conducted on aromatic and aliphatic ketones. The development of the ketones' homologation to the carboxylic acid and the use of the Darzens reaction in converting these ketones to epoxynitriles are described.

Research has been conducted on the ethylene glycol bis(tropane-3-carboxylate) epimers. The synthesis of these epimers has been carried out via the conversion of 3-tropinone into the enol triflate and the details are reported.

Research has been conducted on the carboxylic esters' boron-mediated aldol reaction. The development of the anti- and syn-selective asymmetric aldol reactions with norephedrine-derived propionate esters is described.

Research has been conducted on the lanthadide (III) salts. Results indicate that these salts catalyze symmetrical diol monoacylation via carboxylic acid anhydrides.

Research has been conducted on amides, sters and carboxylic acids. The reduction of these compounds with trialkylsilanes has been carried out via the use of triruthenium carbonyl cluster with the bridging acenaphthylene ligand as a catalyst.

The authors review the publications on organophosphorus compounds. The topics of interest include structure, bonding and nucleophilic reactivity of o-lodosylcarboxylates, and modeling nerve agents.

Research has been conducted on the valence-delocalized diiron(II,III) complexes. The spectroscopic properties, molecular structure and synthesis of these complexes are reported.

Research has been conducted on Ser-Pro and Pro-Ser dipeptides. The proline residue of these dipeptides has been replaced by 7-azabicyclo[2.2.1]-heptane-1-carboxylic acid and the results of the new dipeptides' conformational analysis in the solid state demonstrate that 7-azabicyclo[2.2.1]-heptane-1-carboxylic acid-Ser sequence includes amide bond.

Research has been conducted on the bicyclic munchnones. The results of the intramolecular cycloaddition of these munchnones derived from 2-substituted-N-acylthiazolidine-4-carboxylic acids' cyclodehydration are presented.

Research has been conducted on the ligands with 6-bromo-2,2'-bipyridine pendant arms attached in 5'-position. Results indicate that the transformation of the bromo to the ester has been carried out via the carboethoxylation reaction which has been promoted by low-valent Pd(0).

Research has been conducted on the Diels-Alder reaction. The route to the spiro[2.4]hepta-4,6-diene-4-carboxylic acid esters and spiro[2.4]hepta-4,6-diene-4-methanol has been developed via the use of the hetero-Diels-Alder reaction and the utilization of these dienes for the rac-10-hydroxycamphor synthesis via carbo-Diels-Alder reaction has been carried out.

Research has been conducted on the diiron(II) complexes supported by the sterically requiring carboxylate ligands. The synthetic routes to these complexes have been developed and are described

Research has been conducted on the tricyclic carboxylic acids with the angular methano group. The synthesis of these acids mimicking carbapenams and carbapenems carried out via the intramolecular cyclopropanation mediated by the iminium ion and trimethylstannylmethyl group is described.

Research has been conducted on the role of histone H2A variants in the chromatic array modification. The structural and functional positioning and heterogeneity of this histone within the nucleosome have been investigated and the details are reported.

Research has been conducted on the cadmium(II) coordination polymer. The synthesis and characterization of this polymers have been performed and the details are reported.

Research has been conducted on the hybrid organic-inorganic polymer which has been produced hydrothermally. The synthesis and the crystal structure of this polymers are described.

Research has been conducted on the divalent metal complexes with the N-(phosphonomethyl)iminodiacetic acid. The synthesis and structural characterization of these complexes have been performed and the details are reported.

Research has been conducted on the tetraphenoxy-substituted diazadibenzoperylenes. The synthesis of these compounds performed from the tetrachloroperylene tetracarboxylic acid bisanhydride via the benzylamine imidization is described.

Research has been conducted on the 6-oxoperhydropyridazine-3-carboxylic acid derivatives. The use of these derivatives as templates for the peptide chain geometry control is described.

Research has been conducted on the Fe (super)II active site in 1- aminocyclopropane-1-carboxylic acid oxidase. The geometric and electronic structure of this active site in aminocyclopropane-1-carboxylic acid oxidase and its interactions with the substrate aminocyclopropane-1-carboxylic acid, cosubstrate ascorbate and activator CO (sub)2 have been examined and are described.

Research has been conducted on the benzenetetracarboxylic dianhydrides derivatives. The wavelength-selective photolysis of these derivatives carried out in nitrogen matrixes and the benzdiynes' generation and decomposition dynamics in the nitrogen matrixes have been investigated and are discussed.

Research has been conducted on the irreversible bacterial enzyme inhibitor, 1-amino-2-methylenecyclopropane-1-carboxylic acid. The synthesis of this radiolabled acid and the results of its incubation with 1-aminocyclopropane-1-carboxylate deaminase are discussed.

Research has been conducted on the tetranuclear complexes. The preparation of these complexes via the reaction of zinc carboxylate and 1,3-bis(dimethylamino)-2-propanol in tetrahydrofuran is described.

Research has been conducted on the vancomycin group's glycopeptide antibiotics. The study of the indole-2-carboxylic acid binding to the antibiotic chlororemomycin carried out in investigating the possibility that this binding would occur with negative cooperativity with respect to the antibiotic dimerization is presented.