Determination of the relative configuration of 5,6,7,8-tetrahydromethanopterin by two-dimensional NMR spectroscopy.

The relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin 1, a coenzyme isolated from methanogenic archaea, has been determined by two-dimensional NMR spectroscopy of N5,N10-methenyl-5,6,7,8-tetrahydromethanopterin 2 to be rel-(6R; 7S; 11R). The complete proton resonance assignment of the pterin moiety of N5,N10-methylene-5,6,7,8-tetrahydromethanopterin 3 is described including the relative stereospecific assignment of the C(14a) methylene protons.     

5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis

Abstract In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H₄MPT) is the carrier of the C₁ unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H₄MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H₄MPT, which is reduced in two subsequent coenzyme F₄₂₀-dependent reactions to 5-methyl-H₄MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H₄MPT-dependent reactions are discussed.     

Identification of 6-acetyl-7-methyl-7,8-dihydropterin as a degradation product of 5,10-methenyl-5,6,7,8-tetrahydromethanopterin

During purification procedures and upon aerobic heating with alkali a green-yellow degradation fluorescent product (GY) was formed from 5,10-methenyl-5,6,7,8-tetrahydromethanopterin, an intermediate in the reduction of CO₂ to methane [J. T. Keltjens, L. Daniels, H. G. Janssen, and G. D. Vogels (1983)Eur. J. Biochem.130, 545–552]. GY was suggested to be a 6-(1-oxo)-7,8-dihydropterin. On the basis of the spectral properties and the results of degradation studies, it was now shown that the structure of GY is 6-acetyl-7-methyl-7,8-dihydropterin. This structure was confirmed by synthesis of the compound and other reference substances.     

Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.     

Stimulation of the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in cell-free extracts of Methanobacterium thermoautotrophicum by the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate

The conversion of formaldehyde to methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum was stimulated up to 10-fold by catalytic amounts of the heterodisulfide (CoM-S-S-HTP) of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. The stimulation required the additional presence of ATP, also in catalytic concentrations. ATP and CoM-S-S-HTP were mutually stimulatory on the methylcoenzyme M formation and it was concluded that the compounds were both involved in the reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase. Micromolar concentrations of benzyl viologen or cyanocobalamin inhibited the formaldehyde conversion; these compounds, however, strongly stimulated the reduction of CoM-S-S-HTP. The results described here closely resemble observations made on the activation and reduction of CO₂ to formylmethanofuran indicating that this step and the reductive activation of the methyltransferase are controlled by some common mechanism.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - MFR methanofuran - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP the heterodisulfide of HS-CoM and HS-HTP - BES 2-bromoethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - CN-Cbl cyanocobalamin - HO-Cbl hydroxycobalamin - HBI 5-hydroxybenzimidazole - DMBI 5,6-dimethylbenzimidazole     

Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum.

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).     

Structural requirements for the modulatory effect of 6-substituted pterins on interleukin 2 receptor binding.

(6R)-5,6,7,8-Tetrahydrobiopterin is produced by stimulated human T lymphocytes, and is known to affect various aspects of interleukin-2-directed T cell proliferation. Using an increased apparent affinity of interleukin 2 receptor to interleukin 2 as a measure of activity, this study explores whether other 6-substituted pterins might have the same effect, and what structural features are necessary for activity. Of the compounds tested, only the T-lymphocyte-derived (6R)-5,6,7,8-tetrahydrobiopterin was active. The diastereomeric (6S)-5,6,7,8-tetrahydrobiopterin was inactive, as were 7,8-dihydrobiopterin, sepiapterin, 5,6,7,8-tetrahydroneopterin, 6,7-dimethyl-5,6,7,8-tetrahydropterin and 6-hydroxymethylpterin. 7,8-Dihydroneopterin and neopterin were also found to be inactive. It follows that neither of these compounds participates in the feedback modulation of IL-2 receptor affinity, although both of them can be detected upon IFN-gamma stimulation of human monocytes/macrophages. A computer-based molecular modelling study of (6R)-5,6,7,8-tetrahydrobiopterin and (6R)-5,6,7,8-tetrahydroneopterin revealed substantial differences in overall shape between the two molecules, with certain features figuring prominently in the low-energy conformers of (6R)-5,6,7,8-tetrahydrobiopterin.     

Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Inhibition of GTP cyclohydrolase I by pterins

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.     

Flavonoids from shoots and roots of Trifolium repens (white clover) grown in presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices

White clover (Trifolium repens) plants were grown in the presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices. Flavones, 4',5,6,7,8-pentahydroxy-3-methoxyflavone and 5,6,7,8-tetrahydroxy-3-methoxyflavone, as well as two flavones 3,7-dihydroxy-4'-methoxyflavone and 5,6,7,8-tetrahydroxy-4'-methoxyflavone never previously reported in plants, were isolated. The known 3,5,6,7,8-pentahydroxy-4'-methoxyflavone, 2',3',4',5',6'-pentahydroxy-chalcone, 6-hydroxykaempferol, 4',5,6,7,8-pentahydroxyflavone and 3,4'-dimethoxykaempferol were also obtained. Analysis of extracts obtained from roots and shoots revealed that the compositions of the flavonoid mixtures varied with growing conditions. Quercetin, acacetin and rhamnetin accumulated in roots of inoculated plants, whereas they were not detected in non-inoculated plants.     

(6R)-5,6,7,8-tetrahydro-L-monapterin from Escherichia coli, a novel natural unconjugated tetrahydropterin

The structure of the major tetrahydropterin in Escherichia coli was determined as (6R)-5,6,7,8-tetrahydro-L-monapterin, i. e. (6R)-2-amino-5,6,7,8-tetrahydro-6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)-one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)-configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R)- and (6S)-hexaacetyl-5,6,7,8-tetrahydro-L-monapterins by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry (LC-MS). (6R)-5,6,7,8-Tetrahydro-L-monapterin is a new unconjugated tetrahydropterin from natural sources.     

New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported.     

Novel thrombin inhibitors incorporating non-basic partially saturated heterobicyclic P1-arginine mimetics

The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetrahydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetrahydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P(1)-arginine side-chain mimetics is described. The binding mode of the most potent candidate in the series co-crystallized with human alpha-thrombin, which exhibited an in vitro K(i) of 140nM and more that 478-fold selectivity against trypsin, is discussed.     

Alkaloids from leaves of Annona salzmannii and Annona vepretorum (Annonaceae)

Phytochemical investigation of the leaves of Annona salzmannii and Annona vepretorum (Annonaceae) led to the identification of seven aporphine alkaloids, anonaine, asimilobine, norcorydine, lanuginosine, liriodenine, lysicamine and oxonantenine, as well as, 1,3,6,6-tetramethyl-5,6,7,8-tetrahydro-isoquinolin-8-one and vomifoliol. Liriodenine, anonaine, asimilobine and norcorydine were found in A. salzmannii, while liriodenine, oxonantenine, lanuginosine, lysicamine, vomifoliol and 1,3,6,6-tetramethyl-5,6,7,8-tetrahydro-isoquinolin-8-one were found in A. vepretorum. All these compounds are being described for the first time in the leaves of A. salzmannii and A. vepretorum. This is the first report of 1,3,6,6-tetramethyl-5,6,7,8-tetrahydro-isoquinolin-8-one in the Annonaceae. The NMR data for oxonantenine, lanuginosine and lysicamine were reviewed.     

Identification of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster

Using reversed-phase high-performance liquid chromatography with electrochemical detection we have demonstrated the occurrence of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster. The former is the first time that has been detected in vivo. The identification has been based on the retention times, hydrodinamic voltagrams and the differential concentration in three strains of Drosophila melanogaster. Compared to the wild type, the Punch2 mutant has diminished levels of both pteridines, whereas Henna-recessive3 lacks completely tetrahydropterin and has increased levels of tetrahydrobiopterin, as expected according to their biochemical lesions.     

Heterocyclic analogues of 2-aminotetralins with high affinity and selectivity for the dopamine D3 receptor.

A novel series of 5,6,7,8-tetrahydroquinazolines, 4,5,6,7-tetrahydroindazoles and 4,5,6,7-tetrahydrobenzothiazoles has been prepared, having high affinity and selectivity for the dopamine D3 receptor. The 4-methoxy-5,6,7,8-tetrahydroquinazoline 6i and 2-amino-4,5,6,7-tetrahydrobenzothiazole 8 proved to be agonists with among the highest D3 receptor affinities and selectivities reported to date.     

Melanogenesis inhibition by tetrahydropterines

There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.     

Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.     

Evidence for aromatic ring reduction in the biodegradation pathwayof carboxylated naphthalene by a sulfate reducing consortium

Naphthalene was used as a model compound in order to study the anaerobic pathway of polycyclic aromatic hydrocarbon degradation. Previously we had determined that carboxylation is an initial step for anaerobic metabolism of naphthalene, but no other intermediate metabolites were identified (Zhang & Young 1997). In the present study we further elucidate the pathway with the identification of six novel naphthalene metabolites detected when cultures were fed naphthalene in the presence of its analog 1-fluoronaphthalene. Results from cultures supplemented with either deuterated naphthalene or non-deuterated naphthalene plus [¹³C]bicarbonate confirm that the metabolites originated from naphthalene. Three of these metabolites were identified by comparison with the following standards: 2-naphthoic acid (2-NA), 5,6,7,8-tetrahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. The presence of 5,6,7,8-tetrahydro-2-NA as a metabolite of naphthalene degradation indicates that the first reduction reaction occurs at the unsubstituted ring, rather than the carboxylated ring. The overall results suggest that after the initial carboxylation of naphthalene, 2-NA is sequentially reduced to decahydro-2-naphthoic acid through 5 hydrogenation reactions, each of which eliminated one double bond. Incorporation of deuterium atoms from D₂O into 5,6,7,8-tetrahydro-2-naphthoic acid suggests that water is the proton source for hydrogenation.