Degradation of 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate in Comamonas acidovorans NBA-10

Comamonas acidovorans NBA-10 was previously shown to degrade 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate. Washed cells, grown on a mixture of 4-nitrobenzoate and ethanol, stoichiometrically produced ammonium and 3,4-dihydroxybenzoate from 4-nitrobenzoate under anaerobic conditions provided ethanol was present. In cell extracts 4-hydroxylaminobenzoate was degraded to ammonium and 3,4-dihydroxybenzoate, but this activity was lost upon dialysis. No requirement for a cofactor was found, but rather reduced incubation conditions were necessary to restore enzyme activity. The 4-hydroxylamino-degrading enzyme was purified and the role of this novel type of enzyme in the degradation of nitroaromatic compounds is discussed.Abbreviation 4-ABA 4-aminobenzoate - 4-NBA 4-nitrobenzoate - 4-HABA 4-hydroxylaminobenzoate - 3,4-diHBA 3,4-dihydroxybenzoate     

Biotransformations Of Fluoroaromatic Compounds: Accumulation Of Hydroxylated Products From 3-Fluorophthalic Acid Using Mutant Strains Of Pseudomonas Testosteroni

Biotransformations of 3-fluorophthalic acid have been investigated using blocked mutants of Pseudomonas testosteroni that are defective in the metabolism of phthalic acid (benzene-1,2-dicar-boxyfic acid). Mutant strains were grown with L-glutamic acid in the presence of 3-fluorophthalic acid as inducer of phthalic acid catabolic enzymes. Products that accumulated in the medium were isolated, purified and identified as the fluoroanalogues of those produced from phthalic acid by the same strains. The previously undescribed fluorochemicals cis-3-fluoro-4,5-dihydro-4,5-dihydroxyphthalic acid (VI) and 3-fluoro-4,5-dihydroxyphthalic acid (VII) have been obtained by biotransformation of 3-fluorophthalic acid, and 3-fluoro-5-hydroxyphthalic acid (X) from (VI) by freeze drying. In addition, samples of 2-fluoro-3,4-dihydroxybenzoic acid (2-fluoroprotocatechuic acid, VIII) and 3-fluoro-4,Sdi-hydroxybenzoic acid (5-fluoroprotocatechuic acid, IX) were obtained with a mutant deficient in the ring-fission enzyme, showing that the fluorine substituent in their precursor substrate (VII) is not recognized by the decarboxylase of the pathway, which shows no preference for which carboxyl group is removed. These studies of 3-fluorophthalic acid catabolism demonstrate the opportunities available for the production of novel fluorochemicals in reasonable yields by microbial transformations.     

Distribution of Nuclease O Inhibitor among Aspergillus Species

Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl₂, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl₂ was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.

These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group.     

Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds

Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed.     

The ligase chain reaction distinguishes hepatitis B virus S-gene variants

Abstract The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.     

Effects of cell entrapment on growth rate and metabolic activity of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on the growth rate and metabolic activity of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different cell entrapment media (alginate, carrageenan and polyvinyl alcohol) and three cell-to-matrix ratios (0.1%, 0.2% and 0.6%, w v⁻¹) were examined. Representative species of betaproteobacteria were Alcaligenes faecalis and Comamonas testosteroni whereas Pseudomonas putida was a gammaproteobacteria species studied. Free (non-entrapped) cells were included in the study for comparative purpose. Results indicated that the entrapment, type of entrapment media, and cell-to-matrix ratio had significant effects on the growth and metabolic activity of major groups of bacteria in wastewater treatment. Polyvinyl alcohol entrapped cells had the highest specific growth and specific substrate utilization rates. Increase of cell-to-matrix ratio (from 0.1% to 0.2% or 0.6%) did not improve the specific growth and specific substrate utilization rates. The relative performances provided by different entrapment media of the three species studied were quite consistent. This study showed that the suitable choices of entrapment media and cell-to-matrix ratio are important but similar for major groups of bacteria in wastewater treatment.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.