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Effect of dietary glycine and benzoate level on benzoate metabolism in mink (Mustela vision), blue fox (Alopex lagopus), and raccoon dog (Nyctereutes procyonoides).

Three 2 x 4 factorial experiments were carried out from August to September with 30 juvenile male mink, 24 raccoon dogs, and 24 blue foxes to investigate the effect of dietary glycine supply (low or high) on the efficiency of these species to excrete hippuric acid with incremental benzoate intake (0, 1, 2, or 4 mmol/kg BW). For mink, two additional treatments with 1 or 2 mmol/kg BW of ethyl benzoate were included. A basal low-glycine diet was formulated to meet the minimum protein requirements of fur animals (30% of ME). This diet was supplemented with 0 or 3 g/kg of glycine, or with 0, 1.0, 2.07, or 4.15 g/kg of sodium benzoate for mink and blue foxes, and with 0 or 4.5 g/kg of glycine and 0, 1.58, 3.17, or 6.34 g/kg of sodium benzoate for raccoon dogs, respectively. Two additional diets with .76 or 1.53 g/kg of ethyl benzoate were made for mink. Fecal and urinary benzoic and hippuric acid excretion were measured for 3 d. The 24-h recovery of [14C]benzoic acid injected intraperitoneally was measured from urine, the liver, and the kidneys. All animals appeared healthy and no clinical signs of benzoate overdose were observed. Dietary benzoate level did not affect ADFI or ADG in any species. Glycine supplementation lowered ADFI in mink. The majority of ingested benzoates were absorbed from the gut (over 95%), except in blue foxes, which excreted 6 to 15% of ingested benzoates in feces with incremental increases in benzoate intake. Urinary free benzoic acid excretion accounted for 10% of the ingested benzoates in blue foxes but less than 5% in mink and raccoon dogs. When benzoate intake was 1 mmol/kg BW, mink, blue foxes, and raccoon dogs excreted 71, 77, and 34% of ingested benzoates as hippuric acid in urine, respectively. With higher benzoate intakes, urinary hippuric acid excretion decreased quadratically with mink to 20%, and linearly with blue foxes and raccoon dogs to 45 and 16%, respectively. The hippuric acid pathway appears to be the principal route of benzoate elimination in the mink and blue fox, whereas, in the raccoon dog, other pathways appear to be more important. In mink, the elimination of ethyl benzoate did not differ from that of sodium benzoate. Because glycine conjugation is the primary route of benzoate elimination, it is recommended that benzoate content in fur animal feeds should not exceed 1 g/kg feed on an as-fed basis.

Animals↗

Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter.

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.

Benzoates↗

Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.

Acyl Coenzyme A↗

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.

Acetates↗

Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.

A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.

Anaerobiosis↗

Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants.

Pseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.

Benzoates↗

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp.

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.

Actinomycetales↗

Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.

The TOL plasmid-specified meta-cleavage pathway for the oxidative catabolism of benzoate and toluates branches at the ring cleavage products of catechols and reconverges later at 2-oxopent-4-enoate or its corresponding substituted derivatives. The hydrolytic branch of the pathway involves the direct formation of 2-oxopent-4-enoate or its derivatives, whereas the oxalocrotonate branch involves three enzymatic steps effected by a dehydrogenase, an isomerase, and a decarboxylase, which produce the same compounds. Evidence is presented which shows that benzoate and p-toluate can, under certain circumstances, be catabolized by the hydrolytic branch. However, in a fully functional pathway, only m-toluate is dissimilated via this branch, and benzoate and p-toluate are catabolized almost exclusively by the oxalocrotonate branch. The biochemical basis of this selectivity was found to reside in the high affinity of the dehydrogenase for ring fission products derived from benzoate and p-toluate and its inability to attack the ring fission product derived from m-toluate. Although isomerization of 4-oxalocrotonate occurs spontaneously in vitro, enzymatic isomerization was found to be essential for effective functioning of this branch of the pathway in vivo.

Benzoates↗

Modelling the interaction of small organic molecules with biomacromolecules. III. Interaction of benzoates with anti-p-(p'-azophenylazo)benzoate antibody.

A novel approach for modelling the biological activity of organic molecules, which requires simultaneous consideration of the influence of all factors (topological, steric, hydrophobic, and electronic) that determine the bioactivity, is used to study the interaction of a series of benzoates with anti-p-(p'-azophenylazo)benzoate antibody. The results obtained suggest that this biological interaction proceeds by a two-step stereospecific mechanism. The first step requires a geometrical correspondence between the benzoates and the cavity in the biomacromolecule, which enables the pharmacophore to come into close contact with the receptor. The second step is the orbitally controlled electronic interaction between the active parts of the benzoates and the antibody. The electronic interaction results from pi-charge transfer from the pharmacophore to the biomacromolecule and from the formation of pi-complexes. A proposed mathematical model for this biological interaction exhibits some statistical advantages over existing models.

Antibodies↗

X-ray structural characterization of anhydrous metronidazole benzoate and metronidazole benzoate monohydrate.

Single crystals of anhydrous metronidazole benzoate and its monohydrate were isolated from the same aqueous solution and studied by X-ray diffraction. Anhydrous metronidazole benzoate gives crystals belonging to the triclinic space group P1 and, at ambient temperature, a = 6.649 (2), b = 8.666(1), c = 11.940(3) A, alpha = 76.70(2)degrees, beta = 76.72(2)degrees, gamma = 87.56(2)degrees, V = 651.6(3) A3, Z = 2, and Rw(F) = 0.053. Metronidazole benzoate monohydrate gives crystals belonging to the triclinic space group P1 and, at ambient temperature, a = 7.544(1), b = 7.990(1), c = 12.329(4) A, alpha = 94.33(2)degrees, beta = 97.40(2)degrees, gamma = 101.36(1)degrees, V = 718.6(3) A3, Z = 2, and Rw(F) = 0.041. Thermomicroscopy, differential scanning calorimetry, and thermogravimetry were used for initial characterization of the title species and to investigate possible phase changes on heating. The crystal structure analyses revealed that the metronidazole benzoate molecule adopts different conformations in the two crystal forms. Crystal cohesion in the anhydrous form is due to van der Waals interactions only, whereas in the monohydrate, there is strong intermolecular hydrogen bonding mediated by water molecules. Computer-generated X-ray powder patterns for the two species are distinctly different and serve as reference for their identification.

Benzoates↗

Optimization of the benzoate-inducible benzoate p-hydroxylase cytochrome P450 enzyme system in Aspergillus niger.

Introduction in the fungus Aspergillus niger of multiple copies of the A. niger bphA gene, encoding the cytochrome P450 enzyme benzoate p-hydroxylase, did not result in increased activities of this enzyme [Gorcom RFM van, et al. Mol Gen Genet (1990) 223: 192-197] probably because of low expression levels of the gene encoding the second component of the microsomal cytochrome P450 enzyme system, cytochrome P450 reductase. For improvement of this and other cytochrome-P450-dependent reactions, A. niger strains were constructed in which the copy number of the A. niger cprA gene (encoding cytochrome-P450 reductase) or the copy numbers of both cprA and the cytochrome-P450-encoding gene were increased. Expression of both genes was controlled by their own transcription control regions. Benzoate p-hydroxylase activity of different transformants was determined in microsomal fractions using a newly developed indirect in vitro assay. In transformants containing multiple copies of both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transformants in which the copy number of only one of the genes was increased. These results clearly indicate the importance of co-expression of cytochrome-P450 reductase for achieving maximal cytochrome P450 activities in cytochrome-P450-overproducing filamentous fungi.

Aspergillus niger↗

Benzoate metabolism in Pseudomonas putida(arvilla) mt-2: demonstration of two benzoate pathways.

Benzoate-grown cells of Pseudomonas putida(arvilla) mt-2 contain both metapyrocatechase and pyrocatechase activities, although the former activity is much higher than that of the latter. A spontaneous mutant deficient in metapyrocatechase and 2-hydroxymuconic semialdehyde hydrolyase, the first two enzymes in the meta-cleavage pathway of the ring of catechol, has been isolated from this strain. This mutant grows well on a minimal medium containing benzoate as a sole carbon source and has the high activity of pyrocatechase. These findings indicate that the strain mt-2 possesses the genetic capacity for enzymes of both the meta- and ortho-cleavage pathways of benzoate degradation, but its phenotypic expression is the meta pathway.

Benzoates↗

Report of carcinogenesis bioassay of sodium benzoate in rats: absence of carcinogenicity of sodium benzoate in rats.

The carcinogenicity of sodium benzoate was examined in Fischer 344 rats. Sodium benzoate was administered in the diet for 18 to 24 months at two dose levels; concentrations of 2% and 1% in the diet, which corresponded to the maximum tolerated dose (MTD) and 1/2 MTD as estimated from the data obtained by 6-week toxicity study. Fifty males and 52 females each were used per group. Controls consisted of 25 male rats and 43 female rats. No adverse clinical signs directly attributable to the compound were observed in treated animals. Differences in the average body weight, and mortality rates between treated and control groups were negligible. The results of the statistical test for dose-related trends were not significant (p < 0.05) Although a variety of tumors occurred among test and control rats of each sex, tumors appearing in treated rats were similar in type and number to those in controls. It was concluded that no evidence of carcinogenicity in rats from sodium benzoate was demonstrated.

Animals↗

Quantitative determination of benzyl benzoate in benzyl benzoate lotion NF.

The development of a simple, shorter and more accurate method than the NF method for the determination of benzyl benzoate in Benzyl Benzoate Lotion NF is discussed. Hydrolyzed benzyl benzoate was measured spectrophotometrically. Interference from other ingredients of the lotion, oleic acid and triethanolamine was almost negligible. The proposed method was completed in approximately 15 minutes, as opposed to the two hours required by the NF procedure.

Benzoates↗

Effect of hydroxyl group substituents on pyran ring on hydrolysis rate of benzoates: 2-tetrahydropyranyl benzoate.

The hydrolysis of 2-tetrahydropyranyl benzoate was followed spectrophotometrically at 240 nm and was first order with respect to the compound, independent of pH, and very sensitivie to solvent polarity and had an isotope effect (kD2O/kH2O) near unity. The pH-independent hydrolysis rate was about 1 X 10(6) times faster than that of the corresponding glucosly benzoate. The results suggest that the presence of hydroxyl groups on the pyran ring has tremendous effect on the hydrolysis rate of these compounds.

Benzoates↗

Investigation of isoprenoid benzoates and naphthoates by reversed-phase liquid chromatography. Isocratic elution characteristics of benzoates and naphthoates of C5-C20 terpenoid alcohols.

Conditions for derivatization of C5-C20 isoprenoid alcohols and the reversed-phase liquid chromatographic properties of the corresponding benzoate and naphthoate esters are described. A non-linear response of log k' to changes in the composition of the mobile phase was observed on a Radial-Pak C18 column. In general, the capacity factors (k') increased with increases in carbon content and degree of saturation in the hydrocarbon chain, and decreased with increases in branching and Z double bond content. Replacement of a single hydrogen in the methyl group at C-3 of geranyl benzoate with fluorine had little effect on k', while the difluoro- and trifuoromethyl derivatives showed regular increases in k'. The large extinction coefficients of the aromatic ester moieties, especially the 2-naphthoates, provide high sensitivity for UV detection.

Alcohols↗

Induction of sexual receptivity by oestradiol benzoate in cyclic female rats: influence of ovarian secretions before injection of oestradiol benzoate.

The ability of cyclic female rats to show sexual receptivity 24 h after an injection of 2 microgram oestradiol benzoate (OB) was lost 24 h after ovariectomy. Exposure of cyclic rats to antioestrogen (nitromophene monocitrate) implants 24 h before ovariectomy and OB treatment prevented the latter from inducing sexual receptivity within 24 h of administration. Treatment of ovariectomized rats with constant release implants filled with an oil solution of 15 microgram oestradiol/ml had no behavioural effect in itself, but prepared the rats to show lordosis 24 h after administration of OB. Progesterone treatment (4 mg) induced sexual behaviour in cyclic rats on days other than that of the oestrous cycle when the rats are normally receptive. Evidence is presented that a lower level of oestradiol stimulation than that present duing pro-oestrus was needed for the induction of sexual receptivity in ovariectomized rats. It is suggested that the low basal level of oestradiol which was present throughout the oestrous cycle was necessary for the induction of sexual receptivity and that an increase in oestradial stimulation served to increase the behavioural sensitivity to progesterone.

Animals↗