The stereospecificity of bacterial external flavoprotein monooxygenases for nicotinamide adenine dinucleotide.

It is known that orcinol hydroxylase shows A-stereospecificity for nicotinamide adenine dinucleotide when the enzyme reaction involves the true substrate, orcinol, but when the reaction is carried out in the presence of pseudosubstrates, this enzyme shows an isotope dependent mixed-type Stereospecificity, the degree of which depends on the uncoupling activity of the employed pseudosubstrate [Ribbons, D. W., Ohta, Y. and Higgins, I. J. (1972) in Miami Winter Symposium, The Molecular Basis of Electron Transport (Schultz, J., and Cameron, B. R., eds.), Vol. 4, pp. 251–274, Academic Press, New York]. In this report, the nicotinamide nucleotide stereospecificity of other external flavoprotein monooxygenases from bacterial sources is presented. All of the monooxygenases examined, namely, melilotate hydroxylase, m-hydroxybenzoate-6-hydroxylase, resorcinol hydroxylase, and salicylate hydroxylase, invariably show A-stereospecificity, as in the case of orcinol hydroxylase with true substrate. Unlike orcinol hydroxylase, however, the stereospecificity of salicylate hydroxylase remains A-stereospecific even with pure pseudosubstrate (i.e., benzoate) regardless of the position of the deuterium at the dihydronicotinamide 4-position. On the basis of the information obtained in this investigation, a general rule pertaining to the stereospecificity of nicotinamide nucleotide enzymes is proposed that all external flavoprotein monooxygenases have A-stereospecificity with respect to nicotinamide adenine dinucleotide.     

Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

The stereochemistry of NADH utilization by the flavoenzyme monooxygenase orcinol hydroxylase.

Ribbons et al. (Ribbons, D.W., Ohta, Y., and Higgins, I.J. (1972) in Molecular Basis of Electron Transport, Miami Winter Symposic Series (Schultz, J., and Cameron, B.F., eds) Vol. 4, pp. 251-274, Academic Press, New York) presented a preliminary report that the flavoenzyme monooxygenase orcinol hydroxylase shows mixed type 4R, 4S stereospecificity with respect to dihydronicotinamide oxidation when resorcinol and m-cresol were used as substrate analogs. With the natural substrate orcinol, 4R chirality was maintained. In kinetic isotope experiments reported here, we demonstrate in fact that orcinol hydroxylase maintains 4R stereospecificity with respect to dihydronicotinamide oxidation with all three substrates, orcinol, resorcinol, and m-cresol. Deuterium and tritium kinetic isotope effects were detected under Vmax conditions with (4R)-[4-2H]-, and (4R)-[4-3H]NADH for all three substrates. No isotope effect was observed with (4S)-[4-2H]NADH and tritium labilization from assays with (4S)-[4-3H]-NADH was negligible in all cases.     

Metabolism of resorcinylic compounds by bacteria: orcinol pathway in Pseudomonas putida.

Enrichment cultures yielded two strains of Pseudomonas putida capable of growth with orcinol (3,5-dihydroxytoluene) as the sole source of carbon. Experiments with cell suspensions and cell extracts indicate that orcinol is metabolized by hydroxylation of the benzene ring followed successively by ring cleavage and hydrolyses to give 2 mol of acetate and 1 mol of pyruvate per mol of orcinol as shown: orcinol leads to 2,3,5-trihydroxytoluene leads to 2,4,6-trioxoheptanoate leads to acetate + acetylpyruvate leads to acetate + pyruvate. Evidence for this pathway is based on: (i) high respiratory activities of orcinol-grown cells towards 2,3,5-trihydroxytoluene; (ii) transient accumulation of a quinone, probably 2-hydroxy-6-methyl-1,4-benzoquinone, during grouth with orcinol; (iii) formation of pyruvate and acetate from orcinol, 2,3,5-trihydroxytoluene, and acetylpyruvate catalyzed by extracts of orcinol, but not by succinate-grown cells; (iv) characterization of the product of oxidation of 3-methylcatechol (an analogue of 2,3,5-trihydroxytoluene) showing that oxygenative cleavage occurs between carbons bearing methyl and hydroxyl substituents; (v) transient appearance of a compound having spectral properties similar to those of acetylpyruvate during 2,3,5-trihydroxytoluene oxidation by extracts of orcinol-grown cells. Orcinol hydroxylase exhibits catalytic activity when resorcinol or m-cresol is substituted for orcinol; hydroxyquinol and 3-methylcatechol are substrates for the ring cleavage enzyme 2,3,5-trihydroxytoluene-1,2-oxygenase. The enzymes of this pathway are induced by growth with orcinol but not with glucose or succinate.     

Degradation of orcinol by Aspergillus niger

Aspergillus niger could utilize orcinol (5-methyl-resorcinol or 3,5-dihydroxytoluene) as the sole source of carbon and energy. In the first step of catabolism A. niger hydroxylates orcinol to form 2,3,5-trihydroxytoluene. Its oxidized form, 2-hydroxy-6-methyl-1,4-benzoquinone, was also formed in the culture medium during growth of this organism. Orcinol-grown cells showed a net increase in the intracellular acetate pool, compared with glucose-grown cells. Cell-free extracts of orcinol-grown cells showed higher activity of orcinol hydroxylase, catechol 1,2-oxygenase, and isocitrate lyase than that of glucose-grown cells. Both orcinol-grown and resorcinol-grown cells exhibit similar respiratory activity on all the substrates checked.     

Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC.

The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.     

Oxidation of orcinol by ceruloplasmin and mixed-ligand copper complexes

Unable to oxidize orcinol (3,5-dihydroxytoluene) under conventional conditions, ceruloplasmin (Cp) catalyzes its oxidation when superoxide radicals are injected into the solution with the aid of a high-voltage generator. The O2-. to oxidized orcinol ratio in solution is close to 2:1. The concentration of hydrogen peroxide, which is the product of the Cp-catalyzed dismutase reaction, is about half that of O2-. No slower than by the native enzyme, orcinol in the presence of O2-. is oxidized by Cp depleted of all its type 2 coppers and partly of type 1 Cu2+. Copper complexes with oxalate and pyrophosphate are able to oxidize orcinol under aerobic conditions, one molecule of oxygen being consumed per each oxidized molecule of orcinol. Both the oxidation of orcinol by Cp and by copper complexes are inhibited by cyanide. Orcinol oxidation seems to be caused by singlet oxygen produced in the Cp-catalyzed dismutase reaction.     

Acid loads induced by the detoxification of plant secondary metabolites do not limit feeding by common brushtail possums (Trichosurus vulpecula)

We fed common brushtail possums artificial diets containing a buffer and the plant secondary metabolite (PSM), orcinol, to test the hypothesis that organic acids, common products of PSM metabolism, limit feeding by common brushtail possums (Trichosurus vulpecula). We introduced several diets containing orcinol and a buffer (urinary alkalising agent) over a course of three experiments. A diet containing 2% orcinol (wet matter) caused possums to reduce their food intake immediately, but feeding returned to normal 1–2 days later. Even though possums excreted strongly acidic urine (pH 5.1) and had perturbed nitrogen metabolism, they maintained their food intake and body mass until the experiment terminated 9 days after the introduction of orcinol. Possums ate 52% less when the basal diet contained 4% orcinol. As expected, the acid loads caused a change in the composition of urinary nitrogen with possums excreting more ammonium than urea and a large amount of unidentified nitrogenous material. Supplementing the diet containing orcinol with buffer neutralised the metabolic acid load and partly restored normal nitrogen metabolism, but did not restore feeding. Also, animals eating orcinol excreted normal amounts of 3-methylhistidine, indicating no increase in muscle protein catabolism. This suggests that a limitation to the rate of detoxification or toxicosis, rather than acid loads, limits the ingestion of acid-inducing PSMs.     

Synthesis and properties of new photoactivable derivatives of tetrodotoxin

Eight lots of reagent-grade phenol from four companies were tested for capacity to interact with Cu2+ to produce an inactivator or inactivators of the transfective RNA obtained from poliovirions; such capacity to interact with Cu2+ is referred to as cofactor activity. Six of the lots showed cofactor activity; two did not. A review of the data on the phenol lots and of the properties of the impurity or impurities conferring cofactor activity suggested that the active impurity(ies) might be a dihydric or trihydric phenol. Commercial catechol, resorcinol, hydroquinone, orcinol and pyrogallol were tested and found active. The activity of hydroquinone was outstandingly high. Upon serial recrystallization, the activity of catechol, hydroquinone, orcinol and pyrogallol remained constant, but the activity of resorcinol decreased markedly, in stepwise fashion, showing the most of the activity of the commercial resorcinol was due to impurity(ies). Each of catechol, hydroquinone, orcinol, pyrogallol, and the commercial resorcinol was shown to react with Cu2+ to produce inactivator(s). The effective target for inactivator(s) was the RNA and not the transfection process. The kinetics of inactivator(s) production varied for the different phenols, and the inactivator activity of the incubated mixture of pyrogallol and Cu2+ was notably labile.     

Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.     

Lichen acids,plant growth inhibitors from Usnea longissima

Eight components, depsides and orcinol derivatives which exhibit growth-inhibitory activity agai.     

Requirement of alkaline hydrolysis for estimating chloroplast RNA using the orcinol reaction

Considerable amounts of orcinol-reacting material other than RNA-ribose were extracted with hot PCA from isolated spinach and broad bean chloroplasts, while cold PCA only incompletely extracted the chloroplast RNA. The procedure utilizing alkaline hydrolysis followed by an orcinol test was found to be appropriate for measuring the chloroplast RNA.     

Genotyping and prenatal assessment of collagen lysyl hydroxylase deficiency in a family with Ehlers-Danlos syndrome type VI.

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.     

A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.     

Isolation and identification of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid from the urine of a patient with sialuria.

N-Acetylneuraminic acid preparations from the urine of a patient with sialuria contain 1--2% of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. This new human sialic acid was isolated by ion-exchange and partition chromatography. The structure has been elucidated by mass spectrometry and confirmed by comparison with the synthetic compound. The properties of this unsaturated sialic acid in the orcinol/Fe3+/HCl and the periodic acid/thiobarbituric acid tests as well as in thin-layer and gas-liquid chromatography are described. It does not react with acylneuraminate pyruvatelyase. The origin of this new human sialic acid is discussed.     

Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose. A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes.     

Interaction with a monoclonal antibody alters the expression of co-operativity by phenylalanine hydroxylase from rat liver.

Phenylalanine hydroxylase purified from rat liver shows positive co-operativity in response to variations in phenylalanine concentration when assayed with the naturally occurring cofactor tetrahydrobiopterin. In addition, preincubation of phenylalanine hydroxylase with phenylalanine results in a substantial activation of the tetrahydrobiopterin-dependent activity of the enzyme. The monoclonal antibody PH-1 binds to phenylalanine hydroxylase only after the enzyme has been preincubated with phenylalanine and is therefore assumed to recognize a conformational epitope associated with substrate-level activation of the hydroxylase. Under these conditions, PH-1 inhibits the activity of phenylalanine hydroxylase; however, at maximal binding of PH-1 the enzyme is still 2-3 fold activated relative to the native enzyme. The inhibition by PH-1 is non-competitive with respect to tetrahydropterin cofactor. This suggests that PH-1 does not bind to an epitope at the active site of the hydroxylase. Upon maximal binding of PH-1, the positive co-operativity normally expressed by phenylalanine hydroxylase with respect to variations in phenylalanine concentration is abolished. The monoclonal antibody may therefore interact with phenylalanine hydroxylase at or near the regulatory or activator-binding site for phenylalanine on the enzyme molecule.     

Studies of short-lived radicals in the gamma-irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography.

An aerated aqueous solution of uridine-5'-monophosphate was gamma-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radical which was the radiolytic product of the trapping reagent.     

一株印楝植物内生真菌Epicoccumsp．次生代谢产物的研究

从印楝植物内生真菌Epicoccum sp.的发酵液中分离得到6个化合物,经波谱数据分析分别鉴定为苔黑酚(1)、4-甲基苔黑酚(2)、苔色酸(3)、对羟基苯乙酸(4)、邻苯二甲酸正丁异丁酯(5)、乙基-β-D-葡萄糖苷(6).以上化合物均为首次从该属真菌中分离得到.