Biodegradation of para-Nitrophenol (PNP) proceeds via two distinct pathways, having 1,2,3-benzenetriol (BT) and hydroquinone (HQ) as their respective terminal aromatic intermediates. Genes involved in these pathways have already been studied in different PNP degrading bacteria. Burkholderia sp. strain SJ98 degrades PNP via both the pathways. Earlier, we have sequenced and analyzed a ~41 kb fragment from the genomic library of strain SJ98. This DNA fragment was found to harbor all the lower pathway genes; however, genes responsible for the initial transformation of PNP could not be identified within this fragment. Now, we have sequenced and annotated the whole genome of strain SJ98 and found two ORFs (viz., pnpA and pnpB) showing maximum identity at amino acid level with p-nitrophenol 4-monooxygenase (PnpM) and p-benzoquinone reductase (BqR). Unlike the other PNP gene clusters reported earlier in different bacteria, these two ORFs in SJ98 genome are physically separated from the other genes of PNP degradation pathway. In order to ascertain the identity of ORFs pnpA and pnpB, we have performed in-vitro assays using recombinant proteins heterologously expressed and purified to homogeneity. Purified PnpA was found to be a functional PnpM and transformed PNP into benzoquinone (BQ), while PnpB was found to be a functional BqR which catalyzed the transformation of BQ into hydroquinone (HQ). Noticeably, PnpM from strain SJ98 could also transform a number of PNP analogues. Based on the above observations, we propose that the genes for PNP degradation in strain SJ98 are arranged differentially in form of non-contiguous gene clusters. This is the first report for such arrangement for gene clusters involved in PNP degradation. Therefore, we propose that PNP degradation in strain SJ98 could be an important model system for further studies on differential evolution of PNP degradation functions.
para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.
Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.
Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.
Pseudomonas sp. strain WBC-3; para-Nitrophenol; PNP degradation; γ-hydroxymuconic semialdehyde dehydrogenase; Catalyzing mechanism
para-Nitrophenol (PNP), a priority environmental pollutant, is hazardous to humans and animals. However, the information relating to the PNP degradation pathways and their enzymes remain limited.
Pseudomonas sp.1-7 was isolated from methyl parathion (MP)-polluted activated sludge and was shown to degrade PNP. Two different intermediates, hydroquinone (HQ) and 4-nitrocatechol (4-NC) were detected in the catabolism of PNP. This indicated that Pseudomonas sp.1-7 degraded PNP by two different pathways, namely the HQ pathway, and the hydroxyquinol (BT) pathway (also referred to as the 4-NC pathway). A gene cluster (pdcEDGFCBA) was identified in a 10.6 kb DNA fragment of a fosmid library, which cluster encoded the following enzymes involved in PNP degradation: PNP 4-monooxygenase (PdcA), p-benzoquinone (BQ) reductase (PdcB), hydroxyquinol (BT) 1,2-dioxygenase (PdcC), maleylacetate (MA) reductase (PdcF), 4-hydroxymuconic semialdehyde (4-HS) dehydrogenase (PdcG), and hydroquinone (HQ) 1,2-dioxygenase (PdcDE). Four genes (pdcDEFG) were expressed in E. coli and the purified pdcDE, pdcG and pdcF gene products were shown to convert HQ to 4-HS, 4-HS to MA and MA to β-ketoadipate respectively by in vitro activity assays.
The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies identified 4-NC, HQ, 4-HS, and MA as intermediates in the degradation pathway of PNP by Pseudomonas sp.1-7. This is the first conclusive report for both 4-NC and HQ- mediated degradation of PNP by one microorganism.
para-Nitrophenol; Catabolism; Hydroquinone pathway; Hydroxyquinol pathway; Pseudomonas
The degradation of p-nitrophenol (PNP) by Moraxella and Pseudomonas spp. involves an initial monooxygenase-catalyzed removal of the nitro group. The resultant hydroquinone is subject to ring fission catalyzed by a dioxygenase enzyme. We have isolated a strain of an Arthrobacter sp., JS443, capable of degrading PNP with stoichiometric release of nitrite. During induction of the enzymes required for growth on PNP, 1,2,4-benzenetriol was identified as an intermediate by gas chromatography-mass spectroscopy (GC-MS) and radiotracer studies. 1,2,4-Benzenetriol was converted to maleylacetic acid, which was further degraded by the beta-ketoadipate pathway. Conversion of PNP to 1,2,4-benzenetriol is catalyzed by a monooxygenase system in strain JS443 through the formation of 4-nitrocatechol, 4-nitroresorcinol, or both. Our results clearly indicate the existence of an alternative pathway for the biodegradation of PNP.
Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.
p-Nitrophenol (PNP) occurs as contaminants of industrial effluents and it is the most important environmental pollutant and causes significant health and environmental risks, because it is toxic to many living organisms. Nevertheless, the information regarding PNP degradation pathways and their enzymes remain limited.
To evaluate the efficacy of the Pseudomonas Putida 1274 for removal of PNP.
P. putida MTCC 1274 was obtained from MTCC Chandigarh, India and cultured in the minimal medium in the presence of PNP. PNP degradation efficiency was compared under different pH and temperature ranges. The degraded product was isolated and analyzed with different chromatographic and spectroscopic techniques.
P. putida 1274 shows good growth and PNP degradation at 37°C in neutral pH. Acidic and alkali pH retarded the growth of P. putida as well as the PNP degradation. On the basis of specialized techniques, hydroquinone was identified as major degraded product. The pathway was identified for the biodegradation of PNP. It involved initial removal of the nitrate group and formation of hydroquinone as one of the intermediates.
Our results suggested that P. putida 1274 strain would be a suitable aspirant for bioremediation of nitro-aromatic compounds contaminated sites in the environment.
p-Nitrophenol; Pseudomonas putida; Hydroquinone; Biodegradation; Bioremediation
Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.
We have isolated two soil bacteria (identified as Arthrobacter aurescens TW17 and Nocardia sp. strain TW2) capable of degrading p-nitrophenol (PNP) and numerous other phenolic compounds. A. aurescens TW17 contains a large plasmid which correlated with the PNP degradation phenotype. Degradation of PNP by A. aurescens TW17 was induced by preexposure to PNP, 4-nitrocatechol, 3-methyl-4-nitrophenol, or m-nitrophenol, whereas PNP degradation by Nocardia sp. strain TW2 was induced by PNP, 4-nitrocatechol, phenol, p-cresol, or m-nitrophenol. A. aurescens TW17 initially degraded PNP to hydroquinone and nitrite. Nocardia sp. strain TW2 initially converted PNP to hydroquinone or 4-nitrocatechol, depending upon the inducing compound.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
Benzenetriol dioxygenase; p-Nitrophenol; Phylogenetic analysis
Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported.
Maleylacetate reductase (EC 18.104.22.168) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P21212, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively.
maleylacetate reductase; Burkholderia sp. strain SJ98
Purine nucleoside phosphorylase (PNP) catalyzes the synthesis and phosphorolysis of purine nucleosides, interconverting nucleosides with their corresponding purine base and ribose-1-phosphate. While PNP plays significant roles in human and pathogen physiology, we are interested in developing PNP as a catalyst for the formation of nucleoside analog drugs of clinical relevance. Towards this aim, we describe the engineering of human PNP to accept 2′,3′-dideoxyinosine (ddI, Videx®) as a substrate for phosphorolysis using a combination of site-directed mutagenesis and directed evolution. In human PNP, we identified a single amino acid, Tyr-88, as a likely modulator of ribose selectivity. RosettaLigand was employed to calculate binding energies for substrate and substrate analog transition state complexes for single mutants of PNP where Tyr-88 was replaced with another amino acid. In parallel, these mutants were generated by site-directed mutagenesis, expressed and purified. A tyrosine to phenylalanine mutant (Y88F) was predicted by Rosetta to improve PNP catalytic activity with respect to ddI. Kinetic characterization of this mutant determined a 9-fold improvement in kcat and greater than 2-fold reduction in KM. Subsequently, we used directed evolution to select for improved variants of PNP-Y88F in Escherichia coli cell extracts resulting in an additional 3-fold improvement over the progenitor strain. The engineered PNP may form the basis for catalysts and pathways for the biosynthesis of ddI.
directed evolution; enzyme design; nucleoside analog; purine nucleoside phosphorylase; Rosetta
Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.
A 2-Chloro-4-nitrophenol (2C4NP) degrading bacterial strain designated as RKJ 800 was isolated from a pesticide contaminated site of India by enrichment method and utilized 2C4NP as sole source of carbon and energy. The stoichiometric amounts of nitrite and chloride ions were detected during the degradation of 2C4NP. On the basis of thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry, chlorohydroquinone (CHQ) and hydroquinone (HQ) were identified as major metabolites of the degradation pathway of 2C4NP. Manganese dependent HQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain RKJ 800 that suggested the cleavage of the HQ to γ-hydroxymuconic semialdehyde. On the basis of the 16S rRNA gene sequencing, strain RKJ 800 was identified as a member of genus Burkholderia. Our studies clearly showed that Burkholderia sp. RKJ 800 degraded 2-chloro-4-nitrophenol via hydroquinone pathway. The pathway identified in a gram negative bacterium, Burkholderia sp. strain RKJ 800 was differed from previously reported 2C4NP degradation pathway in another gram-negative Burkholderia sp. SJ98. This is the first report of the formation of CHQ and HQ in the degradation of 2C4NP by any gram-negative bacteria. Laboratory-scale soil microcosm studies showed that strain RKJ 800 is a suitable candidate for bioremediation of 2C4NP contaminated sites.
A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD+, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.
The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5′-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5′ end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5′ double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5′ end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5′ fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.
A study was conducted of possible reasons for acclimation of microbial communities to the mineralization of organic compounds in lake water and sewage. The acclimation period for the mineralization of 2 ng of p-nitrophenol (PNP) or 2,4-dichlorophenoxyacetic acid per ml of sewage was eliminated when the sewage was incubated for 9 or 16 days, respectively, with no added substrate. The acclimation period for the mineralization of 2 ng but not 200 ng or 2 micrograms of PNP per ml was eliminated when the compound was added to lake water that had been first incubated in the laboratory. Mineralization of PNP by Flavobacterium sp. was detected within 7 h at concentrations of 20 ng/ml to 2 micrograms/ml but only after 25 h at 2 ng/ml. PNP-utilizing organisms began to multiply logarithmically after 1 day in lake water amended with 2 micrograms of PNP per ml, but substrate disappearance was only detected at 8 days, at which time the numbers were approaching 10(5) cells per ml. The addition of inorganic nutrients reduced the length of the acclimation period from 6 to 3 days in sewage and from 6 days to 1 day in lake water. The prior degradation of natural organic materials in the sewage and lake water had no effect on the acclimation period for the mineralization of PNP, and naturally occurring inhibitors that might delay the mineralization were not present. The length of the acclimation phase for the mineralization of 2 ng of PNP per ml was shortened when the protozoa in sewage were suppressed by eucaryotic inhibitors, but it was unaffected or increased if the inhibitors were added to lake water.(ABSTRACT TRUNCATED AT 250 WORDS)
A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.
A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-O-dimethyl)-β-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75°C) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes.
The crystallization and preliminary crystallographic analysis of a para-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4 are presented.
Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P212121, with unit-cell parameters a = 54.47, b = 77.56, c = 209.17 Å, and diffracted to 2.24 Å resolution.
para-nitrophenol 4-monooxygenase; Pseudomonas putida DLL-E4
The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to β-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.
Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.
Pseudomonas strains capable of mineralizing 2,4-dichlorophenol (DCP) and p-nitrophenol (PNP) in culture media were isolated from soil. One DCP-metabolizing strain mineralized 1.0 and 10 micrograms of DCP but not 2.0 to 300 ng/ml in culture. When added to lake water containing 10 micrograms of DCP per ml, the bacterium did not mineralize the compound, and only after 6 days did it cause the degradation of 1.0 microgram of DCP per ml. The organism did not grow or metabolize DCP when inoculated into sterile lake water, but it multiplied in sterile lake water amended with glucose or with DCP and supplemental nutrients. Its population density declined and DCP was not mineralized when the pseudomonad was added to nonsterile sewage, but the bacterium grew in sterile DCP-amended sewage, although not causing appreciable mineralization of the test compound. Addition of the bacterium to nonsterile soil did not result in the mineralization of 10 micrograms of DCP per g, although mineralization was evident if the inoculum was added to sterile soil. A second DCP-utilizing pseudomonad failed to mineralize DCP when added to the surface of sterile soil, although activity was evident if the inoculum was mixed with the soil. A pseudomonad able to mineralize 5.0 micrograms of PNP per ml in culture did not mineralize the compound in sterile or nonsterile lake water. The bacterium destroyed PNP in sterile sewage and enhanced PNP mineralization in nonsterile sewage. When added to the surface of sterile soil, the bacterium mineralized little of the PNP present at 5.0 micrograms/g, but it was active if mixed well with the sterile soil.(ABSTRACT TRUNCATED AT 250 WORDS)
A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5′-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3′-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3′ end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3′-to-5′ exonuclease. The finding of impediments to 3′-to-5′ degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
The kinetics of simultaneous mineralization of p-nitrophenol (PNP) and glucose by Pseudomonas sp. were evaluated by nonlinear regression analysis. Pseudomonas sp. did not mineralize PNP at a concentration of 10 ng/ml but metabolized it at concentrations of 50 ng/ml or higher. The Ks value for PNP mineralization by Pseudomonas sp. was 1.1 micrograms/ml, whereas the Ks values for phenol and glucose mineralization were 0.10 and 0.25 micrograms/ml, respectively. The addition of glucose to the media did not enable Pseudomonas sp. to mineralize 10 ng of PNP per ml but did enhance the degradation of higher concentrations of PNP. This enhanced degradation resulted from the simultaneous use of glucose and PNP and the increased rate of growth of Pseudomonas sp. on glucose. The Monod equation and a dual-substrate model fit these data equally well. The dual-substrate model was used to analyze the data because the theoretical assumptions of the Monod equation were not met. Phenol inhibited PNP mineralization and changed the kinetics of PNP mineralization so that the pattern appeared to reflect growth, when in fact growth was not occurring. Thus, the fitting of models to substrate depletion curves may lead to erroneous interpretations of data if the effects of second substrates on population dynamics are not considered.
We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP). The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography. The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN). In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (Km = 2 and 105 microM and kcat = 0.76 and 1.72 s-1 for PNP and PMP, respectively) and produces hydrogen peroxide. Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low kcat turnover number. Comparison of kcat/Km values suggests that PNP rather than PMP is the in vivo substrate of E. coli PdxH oxidase. In contrast, the eukaryotic enzyme has similar kcat/Km values for PNP and PMP and seems to act as a scavenger. E. coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 microM, respectively. Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence. Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E. coli growing exponentially in minimal medium plus glucose at 37 degrees C. Thus, E. coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis.