Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase–prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.     

Stereospecific enzymes in the degradation of aromatic compounds by pseudomonas putida

Two reactions in the catabolism of catechol by meta-fission, namely, hydration of 2-oxopent-4-enoate (vinylpyruvate) and aldol fission of the product, are catalyzed by stereospecific enzymes. The absolute configuration of this hydration product was shown to be l(S)-4-hydroxy-2-oxopentanoate. Vinylpyruvate hydratase, purified almost to homogeneity, had a molecular weight of about 287,000 and was dissociated in sodium dodecyl sulfate, without prior treatment with mercaptoethanol, into a species with an approximate molecular weight of 28,000. The hydratase was highly specific for its substrates; thus, although 2-oxo-cis-hex-4-enoate was also hydrated, structurally similar compounds such as the trans isomer, vinylacetic and crotonic acids, and the ring-fission products of catechol and methylcatechols were not attacked. Vinylpyruvate hydratase was activated by Mn(2+) ions. On the basis of these observations, a mechanism is proposed which closely resembles that for 4-hydroxy-2-oxopentanoate aldolase. A possible evolutionary connection between functionally related, divalent cation-activated hydro-lyases and aldolases is discussed. It was also demonstrated that l-(S)-4-hydroxy-2-oxohexanoate is the biologically active enantiomer of this hydroxy acid.     

Population of the triplet states of bacteriorhodopsin and of related model compounds by intramolecular energy transfer

In variance with chlorophyll-based photosynthetic pigments, the triplet states of rhodopsins, either visual or photosynthetic, have not been observed experimentally. This is due to the ultrafast crossing from S1 to S0, which effectively competes with intersystem crossing to the triplet (T1) state. In order to populate T1 indirectly, laser photolysis experiments are performed with model protonated Schiff bases of retinal in solution, in which both inter- and intramolecular energy transfer to the polyene chromophore are carried out from an appropriate triplet energy donor. The experiments are then extended to bacteriorhodopsin (bR) by detaching the native retinal chromophore from the protein-binding site and replacing it by an analogous (synthetic) protonated Schiff base polyene, attached in a nonconjugated way to a naphthone triplet donor. Pulsed laser excitation of the latter moiety led, for the first time, to the observation of the triplet state of a rhodopsin. Possible locations and roles of the T1 state in bR and in visual pigments are discussed briefly.     

Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (epsilon) of -1.5 and -3.9, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic epsilon (epsilon(intrinsic)) were calculated. A comparison of epsilon(intrinsic) at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific epsilon elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of epsilon found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average epsilon if no fractionation factor is available for single compounds.     

Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45. A highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups.

Hydroxylaminobenzene mutase is the enzyme that converts intermediates formed during initial steps in the degradation of nitrobenzene to a novel ring-fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechanism of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap-SP, Hitrap-Q and Cu(II)-chelating chromatography was used to partially purify the enzyme from an Escherichia coli clone. The partially purified enzyme was eluted in the void volume of a Superose-12 gel-filtration column even in the presence of 0.05% SDS in 25 mM Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was carried out in 18O-labeled water, the product did not contain 18O, as determined by GC-MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate.     

Induction of phenylalanine ammonia lyase and pisatin by photosensitive psoralen compounds

The psoralen compounds, xanthotoxin and 4,5′, 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5′,8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5′,8-trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5′,8-Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed.     

Bacterial sarcosine oxidase: comparison of two multisubunit enzymes containing both covalent and noncovalent flavin

Sarcosine oxidase was purified to homogeneity from Corynebacterium sp. P-1, a soil organism isolated by a serial enrichment technique. The enzyme contains 1 mol of noncovalently bound flavin [flavin adenine dinucleotide (FAD)] plus 1 mol of covalently bound flavin [8 alpha-(N3-histidyl)-FAD] per mole of enzyme (Mr 168,000). The two flavins appear to have different roles in catalysis. The enzyme has an unusual subunit composition, containing four dissimilar subunits (Mr 100,000, 42,000, 20,000, and 6000). The same subunits are detected in Western blot analysis of cell extracts prepared in the presence of trichloroacetic acid, indicating that the subunits are a genuine property of the enzyme as it exists in vivo. The presence of both covalent and noncovalent flavin in a single enzyme is extremely unusual and has previously been observed only with a sarcosine oxidase from a soil Corynebacterium isolated in Japan. The enzymes exhibit many similarities but are distinguishable in electrophoretic studies. Immunologically, the enzymes are cross-reactive but not identical. The results indicate that the synthesis of a sarcosine oxidase containing both covalent and noncovalent flavin is not a particularly unusual event in corynebacteria.     

Hydroxylation of aromatic compounds by reduced nicotinamide-adenine dinucleotide and phenazine methosulphate requires hydrogen peroxide and hydroxyl radicals, but not superoxide.

1. A mixture of NADH and phenazine methosulphate hydroxylates aromatic compounds at acidic pH values. 2. Hydroxylation is inhibited by catalase and by scavengers of the hydroxyl radical (-OH) but not by superoxide dismutase. 3. It is concluded that neither O2 leads to nor HO2- is sufficiently reactive to hydroxylate aromatic rings.     

Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice

The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohol groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. Lipase (EC 3.1.1.3) did not hydrolyze compounds that contained more than three ester groups. Compounds containing four and five ester groups were hydrolyzed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolyzed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolyzed.     

Production of hexanal from hydrolyzed sunflower oil by lipoxygenase and hydroperoxid lyase enzymes

Hexanal was produced from hydrolyzed sunflower oil in two steps: 1) 13-hydroperoxy-9-(Z),11(E)-octadecadienoic acid (13-HPOD) was formed from linoleic acid (100 mM) by soybean lipoxygenase-1 isoenzyme (Lox-1) with O₂, the reaction resulted 68.7 mM 13-HPOD with a yield of 72%. 2) 13-HPOD (15 mM) was cleaved by spinach leaf hydroperoxide lyase resulting 8.2 mM hexanal (54% yield). Hexanal was isolated from the reaction mixture by repeated steam distillation.     

Sulfhydryl groups in glycolipid transfer protein: formation of an intramolecular disulfide bond and oligomers by Cu2+-catalyzed oxidation

Glycolipid transfer protein (GLTP) purified from pig brain facilitates the transfer of various glycolipids between lipid bilayers. Purified GLTP migrates as two bands of different mobility in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The slower component and the faster component constituted about 80% and about 15%, respectively, of purified GLTP. Treatment of GLTP with 45 microM CuSO4 resulted in a decrease in the slower component, an increase in the faster component, and the formation of oligomeric components. The faster and oligomeric components were quantitatively converted to the slower component by reduction with 2% 2-mercaptoethanol in the presence of 1% SDS. The formation of oligomeric components was enhanced by increasing the concentration of CuSO4 to 450 microM and 4.5 mM. Oxidation of GLTP catalyzed by CuSO4 resulted in a decrease in the transfer activity and an increase in the apparent binding affinity of GLTP to 1-O-(beta-D-galactopyranosyl)-N-[10-(1-pyrenyl)decanoyl]-D-erythro- sphingosine (PyrGalCer). The oligomeric components and the monomeric components were isolated by chromatography on a Sephadex G-75 column. It was found that GLTP in fractions enriched with the monomeric components had very high transfer activity and is responsible for most of the transfer activity in the oxidized GLTP. Treatment of GLTP with 1.27 mM HgCl2 resulted in a formation of components unresolvable on SDS-PAGE and also resulted in a reduction of the transfer activity to one-third. However, no obvious change in the binding affinity of GLTP to PyrGalCer was observed by HgCl2 treatment. Treatment with 2-mercaptoethanol restored the activity of GLTP inactivated by HgCl2, whereas the activity inactivated by CuSO4 was not restored by treatment with 2-mercaptoethanol. These results suggest that the transfer activity depends on the turnover rate of the GLTP-PyrGalCer complex which is affected by modification of sulfhydryl groups of GLTP. The sulfhydryl group content of GLTP was estimated by the use of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). A value of 2.2 mol sulfhydryl groups per mol of GLTP was found in the presence of 0.5% SDS and one sulfhydryl group in a GLTP molecule was very rapidly oxidized in the native state, from which it is assumed that the slower component contains three sulfhydryl groups per GLTP molecule and the faster component contains one sulfhydryl group and one disulfide bond per GLTP molecule.     

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.     

Role of aliphatic and phenolic hydroxyl groups in uranyl complexation by humic substances

Relativistic density functional calculations of uranyl complexes with alcohols were carried out to study how phenolic and aliphatic hydroxyl groups of humic substances may contribute to uranyl complexation by humic substances. According to recent experimental work, blocking of phenolic OH groups decreases the loading capacity, but has no effect on the key geometric parameters of uranyl humate complexes. This can be understood on the basis of our calculations which showed uranium-oxygen distances to be very similar for complexes with rather different types of O-donor ligands, with average U-O_eq ∼ 237 pm for fivefold coordinated uranyl (VI) complexes, both for O⁻ and OH functional groups. Uranyl complexation by alcohol moieties seems unlikely at environmental conditions as a high pH is required for the deprotonation of these groups; we confirm an alternative complexation mechanism that overcomes the ligand deprotonation problem. Similarities in structures and energetic suggest that complexes of both aliphatic and phenolic alcoholates may well contribute in comparable fashion to the complexation of uranyl by humic acids.     

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.     

Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers

The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ. The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity. We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer. Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit. By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated. In contrast, at 38 degrees C, it was the whole tetramer that was inactivated. beta-Galactosidase cannot have two different functional units depending on temperature. The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodegradation of aromatic compounds and TCE by a filamentous bacteria-dominated consortium

The Michaelis-Menten biodegradation kinetics (k and Ks) of aromatic compounds and trichloroethene (TCE) by an aerobic enrichment culture grown on phenol and dominated by a unique filamentous bacterium were measured. The average k and Ks values for phenol, benzene (B), toluene (T), ethylbenzene (E), o-xylene (oX), p-xylene (pX), naphthalene and TCE in g per g VSS-d and mg L-1 were 5.72 and 0.34, 1.20 and 0.51, 2.09 and 0.47, 0.77 and 0.23, 0.61 and 0.16, 0.73 and 0.23, 0.17 and 0.18, and 0.16 and 0.18, respectively. Significant variability in these measured kinetics was noted between tests conducted over the 5-month period during which the fed-batch culture with a 5-day solids retention time was maintained; the coefficient of variation of the k and Ks values ranged from 11–43% and 4–50%, respectively. This variation was significantly greater than the method measurement error on a given date. Degradation of BTEoXpX mixtures could be described by a basic competitive inhibition model.Batch tests during which the culture was fed individual BTEX compounds showed the culture grew poorly on the xylenes and had poor subsequent xylene degradation rates. This work indicates the potential to simultaneously treat a mixture of volatile organic compounds using this consortium, and the ability to predict the mixture biodegradation rates on the basis of the individual compound biodegradation kinetics.     

Oxidation of methyl groups by grass grubs and vertebrate liver enzymes

1. Oxidation rates of p-nitrotoluene, p-acetamidotoluene and p-toluidine by intact grass grubs and vertebrate liver preparations were measured. 2. The effect of p-substitution in increasing the rate of conversion of the methyl into a carboxy group was in the order acetamido> nitro[unk] amino in mice and grass grubs. 3. Rates of oxidation of the N-methyl group in some alkylaryl N-methylcarbamates was measured and the effect of ring substituents in increasing the rate was in the order hydrogen> o-methyl or o-isopropyl> p-methyl or p-isopropyl> m-methyl or m-isopropyl. 4. Rates of oxidation of the N-methyl groups were similar to those of the p-substituted toluenes.