Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.     

Pollination biology of two species ofKielmeyera (Guttiferae) from Brazilian cerrado vegetation

The pollination biology and breeding systems ofKielmeyera coriacea andK. speciosa, two sympatric woody species common in the cerrado vegetation of C. Brazil, were studied. Both species have similar nectarless, polystemonous Papaver-type flowers which are visited by a similar spectrum of insects, though they bloom in different seasons and are thus phenologically isolated. Large carpenter bees seem to be the most important pollinators and these and other bees effect buzz pollen retrieval despite the fact that anthers are not poricidal. Both species ofKielmeyera possess strong xenogamous breeding systems. The presence of staminate flowers and andromonoecy inK. coriacea, as well as the longevity ofK. speciosa flowers are discussed as alternative strategies to improve pollination success and reproductive efficacy.     

DL-4-hydroxyphenylglycine catabolism in Pseudomonas putida MW27

Thirteen bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. Seven strains transaminated only the D-enantiomer while the other six isolates transaminated both enantiomers of 4-hydroxyphenylglycine. One of the six strains utilizing both enantiomers was characterized as a Pseudomonas putida. This strain, MW27, employed two enantioselective transaminases, to catalyze the initial step in the metabolism of DL-4-hydroxyphenylglycine. The product of the transamination, 4-hydroxyphenylglyoxylate, was further metabolized via 4-hydroxybenzaldehyde and 4-hydroxybenzoate to protocatechuate. Preliminary results indicate that both transaminases are co-ordinately synthesized together with the 4-hydroxyphenylglyoxylate decarboxylase and the NADP⁺-dependent 4-hydroxybenzaldehyde dehydrogenase.     

The in vivo construction of 4-chloro-2-nitrophenol assimilatory bacteria

Pseudomonas sp. N31 was isolated from soil using 3-nitrophenol and succinate as sole source of nitrogen and carbon respectively. The strain expresses a nitrophenol oxygenase and can use either 2-nitrophenol or 4-chloro-2-nitrophenol as a source of nitrogen, eliminating nitrite, and accumulating catechol and 4-chlorocatechol, respectively. The catechols were not degraded further. Strains which are able to utilize 4-chloro-2-nitrophenol as a sole source of carbon and nitrogen were constructed by transfer of the haloaromatic degrading sequences from either Pseudomonas sp. B13 or Alcaligenes eutrophus JMP134 (pJP4) to strain N31. Transconjugant strains constructed using JMP134 as the donor strain grew on 3-chlorobenzoate but not on 2,4-dichlorophenoxyacetate. This was due to the non-induction of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase. Transfer of the plasmid from the 2,4-dichlorophenoxyacetate negative transconjugant strains to a cured strain of JMP134 resulted in strains which also had the same phenotype. This indicates that a mutation has occurred in pJP4 to prevent the expression of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase.     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.