Degradation of 4-Fluorobiphenyl by Mycorrhizal Fungi as Determined by 19F Nuclear Magnetic Resonance Spectroscopy and 14C Radiolabelling Analysis

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using ¹⁹F nuclear magnetic resonance (NMR) spectroscopy in combination with ¹⁴C radioisotope-detected high-performance liquid chromatography (¹⁴C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. ¹⁴C-HPLC profiles indicated that there were four major biotransformation products, whereas ¹⁹F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4′-ol and 4-fluorobiphen-3′-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.     

Biotransformation of fluorobiphenyl by Cunninghamella elegans

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by ¹⁹F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.     

Conformational changes in the human estrogen receptor observed by (19)F NMR

The (19)F NMR spectra of the 5F-Trp labeled glutathione-S-transferase fusion protein with residues 282-595 of the human estrogen receptor show that there is a distinct conformational change in the protein when estradiol is added to the unliganded protein. Our studies show the empty receptor to have more conformational flexibility than the liganded form. This study shows the applicability of (19)F NMR to study conformational change in large protein systems.     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

Testosterone 1 beta-hydroxylation by human cytochrome P450 3A4.

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6 beta-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6 beta- and 2 beta-hydroxy products, was identified as 1 beta-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of approximately 6 microg of this product. Mass spectrometry established the formula as C(19)H(29)O(3) (MH(+) 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the beta-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1 alpha-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.     

Reductive formation of carbon monoxide from CCl4 and FREONs 11, 12, and 13 catalyzed by corrinoids

In an earlier publication, we reported that corrinoids catalyze the sequential reduction of CCl4 to CHCl3, CH2Cl2, CH3Cl, and CH4 with titanium(III) citrate as electron donor [Krone, U. E., Thauer, R. K., & Hogenkamp, H. P. C. (1989) Biochemistry 28, 4908-4914]. However, the recovery of these products was less than 50%, indicating that other products were formed. We now report that, under the same experimental conditions, CCl4 is also converted to carbon monoxide. These studies were extended to include FREONs 11, 12, 13, and 14. Corrinoids were found to catalyze the reduction of CFCl3, CF2Cl2, and CF3Cl to CO and, in the case of CFCl3, to a lesser extent, to formate. CF4 was not reduced. The rate of CO and formate formation paralleled that of fluoride release. Both rates decreased in the series CFCl3, CF2Cl2, CCl4, and CF3Cl. The reduction of CFCl3 gave, in addition to CO and formate, CHFCl2, CH2FCl, CH3F, C2F2Cl2, and C2F2Cl4. The product pattern indicates that the corrinoid-mediated reduction of halogenated C1-hydrocarbons involves the intermediacy of dihalocarbenes, which may be a reason why these compounds are highly toxic for anaerobic bacteria.     

Rosavin as a product of glycosylation by Rhodiola rosea (roseroot) cell cultures

The paper discusses glycosylation of trans-cinnamyl alcohol to obtain the biologically active compound rosavin and possibly other cinnamoylglycosides. Cell suspension cultures of Rhodiola rosea were established from callus of leaf origin cultured under light in a modified Murashige and Skoog medium. Under these conditions, no rosavin was formed. However, when trans-cinnamyl alcohol (2.5 mM; in MeOH) was added to the medium, after 72 h incubation cells transformed over 90% of the cinnamyl alcohol into a number of unidentified products. The structure of potential rosavin accumulated in intracellular spaces was elucidated as [3-phenyl-2-propenyl-O-(6'-O-α-L-arabinopyranosyl) -β-D-glucopyranoside] by means of chemical and spectral analysis using TLC, HPLC, UV, LSIMS and NMR methods. Rosavin yields of 0.03–1.01% dry weight were obtained. The actual amount depended on the cell strain cultured and the biotransformation period. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and ¹⁹F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The ¹⁹F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

The assimilation of organic hazardous wastes by municipal solid waste landfills

Summary Co-disposal of 12 compounds representing major organic classes (aromatic hydrocarbons, halogenated hydrocarbons, pesticides, phenols, and phthalate esters) with shredded municipal solid waste was tested using a laboratory-scale column and pilot-scale lysimeter to characterize transport and transformation phenomena including sorption, volatilization and bioassimilation. Leachate and gases emitted from the lysimeters were examined for identifiable products of biotransformation. The results of this investigation provided a mechanistic evaluation of the attenuating and assimilative capacity of municipal solid waste landfills for specific organic compounds. Physical/chemical organic compound characteristics were related to refuse characteristics and composition to predict compound fate. Such knowledge is useful in developíng landfill management and operational strategies consistent with the need for control of pollutant releases.     

Prospects for in vivo NMR methods in xenobiotic research in plants

The application of non-invasive nuclear magnetic resonance (NMR) methods in xenobiotic research is reviewed in relation to: (i) the characterisation of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection of xenobiotics and their degradation products in vivo; and (iii) the spatial localisation of xenobiotics and their derivatives at the subcellular and tissue levels. Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy is shown to be informative when a xenobiotic perturbs metabolic pathways that are accessible to the technique, and it is useful for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. The successful application of 19F NMR to the analysis of plant tissue extracts also suggests that in vivo 19F NMR spectroscopy may have a role in biotransformation studies of fluorinated xenobiotics. In contrast NMR imaging techniques have been little used for xenobiotic research in plants, and while the method has been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants, the potential use of high resolution 1H and 19F NMR imaging for mapping agrochemicals in tissues is still in its infancy.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines.     

19F NMR investigations of the catalytic mechanism of phosphoglucomutase using fluorinated substrates and inhibitors.

The complexes of phosphoglucomutase with a number of fluorinated substrate analogues have been investigated by 19F NMR and the effects of the binding of Li+ and Cd2+ to these complexes determined. Very large downfield chemical shift changes (-14 to -19 ppm) accompanied binding of the inhibitors 6-deoxy-6-fluoro-alpha-D-glucopyranosyl phosphate and alpha-glucosyl fluoride 6-phosphate to the phosphoenzyme. Smaller shift changes were observed for ligands substituted with fluorine at other positions. Addition of Li+ to enzyme/fluorinated ligand complexes caused a 10(2)- to 10(3)-fold decrease in ligand dissociation constants as witnessed by the change from intermediate to slow-exchange conditions in the NMR spectra. Measurement of the 19F NMR spectra of complexes of the Li(+)-enzyme with each of the fluoroglucose 1-phosphates and 6-phosphates has provided some insight into the environment of each of these fluorines (thus also parent hydroxyls) in each of the complexes. Results obtained argue strongly against a single sugar binding mode for the glucose 1- and 6-phosphates. Two enzyme-bound species were detected in the 19F NMR spectra of the complexes formed by reaction of the Cd(2+)-phosphoenzyme complex with the 2- and 3-fluoroglucose phosphates. These are tentatively assigned as the fluoroglucose 1,6-bisphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of the 4-fluoroglucose phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone structure confirmation and side chain conformation refinement of a bradykinin mimic BKM-824 by comparing calculated (1)H, (13)C and (19)F chemical shifts with experiment

Calculated and experimental (1)H, (13)C and (19)F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava(1)-Igl(2)-Ser(3)-DF5F(4)-Oic(5)-Arg(6)] (Ava=5-aminovaleric acid, Igl=alpha-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)-octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947-955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6-31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6-311G** basis set. There is an intramolecular hydrogen bond at Ser(3) in the optimized structures. The experimental (13)C chemical shifts at five C(alpha) positions as well as at the Cbeta, Cgamma and Cdelta position of Ava(1), which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H(beta) chemical shifts in Igl(2) shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the chi(1) angle in DF5F(4) suggest that chi(1)180 degrees. The calculated (1)H and (13)C chemical shifts are in good agreement with experiment at the rigid residue Oic(5). None of the models gives accurate results for Arg(6), presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.     

Progesterone side-chain degradation by some species ofAspergillus flavus group

Seventy isolates belonging to 6 species and one variety of A. flavus group were shown to degrade the progesterone side-chain to yield delta 4-androstene-3,17-dione and testosterone. The isolates of five species (A. flavo-furcatis, A. flavus, A. oryzae, A. parasiticus and A. tamarii) possessed enzyme systems catalyzing the opening of ring D and formed testololactone as final steroid metabolite in addition to their ability to produce the above mentioned two products. 11 beta-Hydroxy-delta 4-androstene-3,17-dione was formed by only A. flavus and A. tamarii while 11 beta-hydroxytestosterone was produced by A. flavo-furcatis, A. parasiticus and A. subolivaceus. The chromatographic resolution of the mixture products obtained (when the selective isolate of each species reacted with 1 g of progesterone) revealed that 60-75% of progesterone was converted into delta 4-androstene-3,17-dione (8-30%), testosterone (7-33%), testololactone (14-37%) and other products (3-40%). The most bioconversion activity was exhibited by A. oryzae, followed by A. parasiticus. The highest values of delta 4-androstene-3,17-dione (30% of added progesterone) and testosterone (33%) were formed by A. flavus var. columnaris while those of testololactone (37%) were produced by A. oryzae. A systematic variation could be observed between the different tested species of A. flavus group with respect to the transformation reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the tested species in this group; this biochemical differentiation may supplement the morphological and other physiological criteria used in the identification of the different species in the A. flavus group.     

Biotransformation of 2-(4-methoxybenzyl)-l-cyclohexanone by cell cultures ofSolatium aviculare

During the search for compounds with insect juvenile hormone activity, the biotransformation of 2-(4-methoxybenzyl)-l-cyclohexanone, of 2-(4-methoxybenzyl)-l-cyclohexanone ethylene acetal and of both isomers of 2-(4-methoxybenzyl)-l-cyclohexanol by plant cells was examined. The compounds were metabolized by cell suspension culture of Solatium aviculare Forst. The reaction conditions were optimized and the metabolic products isolated and identified. A scheme of biotransformation pathway has been proposed.     

Chloroform mineralization by toluene-oxidizing bacteria.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.