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Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2-threefold higher titres of PDCA production from lignocellulose (200-287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.

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Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

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Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.

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OBJECTIVE: Oxygen scavenging systems are routinely used during single-molecule imaging experiments to improve fluorescent dye stability. Previous work has shown nuclease contamination in the commonly used oxygen scavenging systems. This study evaluates the potential for nuclease contamination in these oxygen scavenging systems. RESULTS: Linear and plasmid DNA was incubated with two different oxygen scavenging systems (1) protocatechuic acid (PCA)-protocatechuate-3,4-dioxygenase (PCD) and (2) glucose-coupled glucose oxidase/catalase (GODCAT). No nucleic acid degradation was observed on single and double-stranded linear DNA and plasmid DNA, indicating the absence of nuclease contamination in these oxygen scavenging systems.

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Protocatechuate 3,4-dioxygenase (P34O), which is isolated from Rhizobium sp. LMB-1, catalyzes the ring cleavage step in the metabolism of aromatic compounds, and has great potential for environmental bioremediation. However, its structure is very sensitive to different environmental factors, which weaken its activity. Immobilization of the enzyme can improve its stability, allow reusability, and reduce operation costs. In this work, the relative molecular mass of the native P34O enzyme was determined to be 500 kDa by gel filtration chromatography on Sephadex G-200, and the enzyme was immobilized onto (3-aminopropyl) triethoxysilane-modified Fe3O4 nanoparticles (NPs) by the glutaraldehyde method. The optimum pH of immobilized and free P34O was unaffected, but the optimum temperature of immobilized P34O increased from 60 to 70 °C, and the thermal stability of immobilized P34O was better than that of the free enzyme and showed higher enzymatic activity at 60 and 70 °C. In addition, with the exception of Fe3+, most metal ions and organic chemicals could not improve the activity of free and immobilized P34O. The kinetic parameters of the immobilized P34O were higher than those of the free enzyme, and immobilized P34O on Fe3O4 NPs could be reused ten times without a remarkable decrease in enzymatic activity.

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A benzoate-degrading archaeal enrichment was developed using sediment samples from Rozel Point at Great Salt Lake, UT. The enrichment degraded benzoate as the sole carbon source at salinity ranging from 2.0 to 5.0 M NaCl with highest rate of degradation observed at 4.0 M. The enrichment was also tested for its ability to grow on other aromatic compounds such as 4-hydroxybenzoic acid (4-HBA), gentisic acid, protocatechuic acid (PCA), catechol, benzene and toluene as the sole sources of carbon and energy. Of these, the culture only utilized 4-HBA as the carbon source. To determine the initial steps in benzoate degradation pathway, a survey of ring-oxidizing and ring-cleaving genes was performed using degenerate PCR primers. Results showed the presence of 4-hydroxybenzoate 3-monooxygenase (4-HBMO) and protocatechuate 3, 4-dioxygenase (3,4-PCA) genes suggesting that the archaeal enrichment might degrade benzoate to 4-HBA that is further converted to PCA by 4-HBMO and, thus, formed PCA would undergo ring-cleavage by 3,4-PCA to form intermediates that enter the Krebs cycle. Small subunit rRNA gene-based diversity survey revealed that the enrichment consisted entirely of class Halobacteria members belonging to the genera Halopenitus, Halosarcina, Natronomonas, Halosimplex, Halorubrum, Salinarchaeum and Haloterrigena. Of these, Halopenitus was the dominant group accounting for almost 91 % of the total sequences suggesting their potential role in degrading oxygenated aromatic compounds at extreme salinity.

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The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L(-1). The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200-250 mg L(-1)) and high (4 g L(-1)) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.

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Previous studies have shown that naturally occurring phytochemicals, indole-3-carbinol, phenethyl isothiocyanate, protocatechuic acid, and tannic acid increased the activity and protein level of hepatic phase II enzymes in animal models. In order to further explore the mechanism of this activity, we investigated the effect of these compounds on the activation of nuclear factor erythroid-2-related factor 2 (Nrf2)-regulated transcription in human hepatocellular carcinoma HepG2 cells. Treatment with all the tested compounds resulted in the translocation from the cytosol and nuclear accumulation of active phosphorylated Nrf2. Furthermore, phenethyl isothiocyanate and indole-3-carbinol increased the transcript and protein levels of GSTA, GSTP, GSTM, GSTT, and NQO1. On the other hand, protocatechuic and tannic acids enhanced only the expression of GSTA, GSTM, and GSTT. The expression of genes encoding antioxidant enzymes CAT, SOD, GR, and GPx was increased after the treatment with all the tested phytochemicals. These results indicate that isothiocyanates/indoles and protocatechuic and tannic acids induce phase II and antioxidant gene expression in HepG2 cells through the Nrf2-Keap1-ARE signaling pathway. Moreover, the results of this study confirmed that the degradation products of glucosinolates are more effective inducers of phase II and antioxidant enzymes than protocatechuic and tannic acids

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Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15µM) and D3 for MhpB (IC50 110µM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the ß-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.

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Phytoestrogens are plant-derived hormonally-active compounds known to cause varied reproductive, immunosuppressive and behavioral effects in vertebrates. In this study, biodegradation of luteolin, a common phytoestrogen, was investigated during incubation with endophytic fungus Phomopsis liquidambari. The optimum concentration of luteolin as sole carbon source supplied in culture was 200 mg L(-1), which allowed 97 and 99 % degradation of luteolin by P. liquidambari in liquid culture and soil conditions, respectively. The investigation of the fungal metabolic pathway showed that luteolin was first decomposed to caffeic acid and phloroglucinol. These intermediate products were degraded to protocatechuic acid and hydroxyquinol, respectively, and then rings were opened by ring-cleavage dioxygenases. Two novel genes encoding the protocatechuate 3,4-dioxygenase and hydroxyquinol 1,2-dioxygenase were successfully cloned. Reverse-transcription quantitative polymerase chain reaction demonstrated that expression levels of mRNA of these two genes increased significantly after P. liquidambari was induced by the intermediate products caffeic acid and phloroglucinol, respectively. These results revealed that P. liquidambari can biodegrade luteolin efficiently and could potentially be used to bioremediate phytoestrogen contamination.

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Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe(3+) to activate O2 and catecholic substrates for reaction. The inability of Fe(3+) to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe(3+) species, and the anhydride-Fe(3+) intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe(2+)-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe(2+) intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

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Protocatechuate 3,4-dioxygenases (P34Os) catalyze the reaction of the ring cleavage of aromatic acid derivatives. It is a key reaction in many xenobiotic metabolic pathways. P34Os characterize narrow substrate specificity. This property is an unfavorable feature in the biodegradation process because one type of pollution is rarely present in the environment. Thus, the following study aimed at the characterization of a P34O from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic carboxylic acids. A total of 3 mM vanillic acid and 4-hydroxybenzoate were completely degraded during 8 and 4.5 h, respectively. When cells of strain KB2 were grown on 9 mM 4-hydroxybenzoate, P34O was induced. Biochemical analysis revealed that the examined enzyme was similar to other known P34Os, but showed untypical wide substrate specificity. A high activity of P34O against 2,4- and 3,5-dihydroxybenzoate was observed. As these substrates do not possess ortho configuration hydroxyl groups, it is postulated that their cleavage could be connected with their monodentate binding of substrate to the active site. Since this enzyme characterizes untypical wide substrate specificity it makes it a useful tool in applications for environmental clean-up purposes.

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Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5 °C and 10 °C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.

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Three diamino, dihetero-phenol ligands were synthesized by sequential Mannich condensations. These ligands were combined with FeCl3 to produce three five-coordinate Fe(III) complexes that are structural models for the enzyme 3,4-PCD. The three Fe(III) complexes were characterized by elemental analysis, single crystal X-ray diffraction studies, UV-vis spectroscopy, and cyclic voltammetry. Combining the Fe(III) complexes with 3,5-di-t-butylcatechol and O2 resulted in oxidative cleavage similar to the function of 3,4-PCD.

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The metabolic pathway of eugenol degradation by thermophilic Geobacillus sp. AY 946034 strain was analyzed based on the lack of data about eugenol degradation by thermophiles. TLC, GC-MS, and biotransformation with resting cells showed that eugenol was oxidized through coniferyl alcohol, and ferulic and vanillic acids to protocatechuic acid before the aromatic ring was cleaved. The cell-free extract of Geobacillus sp. AY 946034 strain grown on eugenol showed a high activity of eugenol hydroxylase, feruloyl-CoA synthetase, vanillate-O-demethylase, and protocatechuate 3,4-dioxygenase. The key enzyme, protocatechuate 3,4- dioxygenase, which plays a crucial role in the degradation of various aromatic compounds, was purified 135-fold to homogeneity with a 34% overall recovery from Geobacillus sp. AY 946034. The relative molecular mass of the native enzyme was about 450 ± 10 kDa and was composed of the non-identical subunits. The pH and temperature optima for enzyme activity were 8 and 60°C, respectively. The half-life of protocatechuate 3,4-dioxygenase at the optimum temperature was 50 min.

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Plant-derived aromatic compounds are important components of the dissolved organic carbon pool in coastal salt marshes, and their mineralization by resident bacteria contributes to carbon cycling in these systems. Members of the roseobacter lineage of marine bacteria are abundant in coastal salt marshes, and several characterized strains, including Sagittula stellata E-37, utilize aromatic compounds as primary growth substrates. The genome sequence of S. stellata contains multiple, potentially competing, aerobic ring-cleaving pathways. Preferential hierarchies in substrate utilization and complex transcriptional regulation have been demonstrated to be the norm in many soil bacteria that also contain multiple ring-cleaving pathways. The purpose of this study was to ascertain whether substrate preference exists in S. stellata when the organism is provided a mixture of aromatic compounds that proceed through different ring-cleaving pathways. We focused on the protocatechuate (pca) and the aerobic benzoyl coenzyme A (box) pathways and the substrates known to proceed through them, p-hydroxybenzoate (POB) and benzoate, respectively. When these two substrates were provided at nonlimiting carbon concentrations, temporal patterns of cell density, gene transcript abundance, enzyme activity, and substrate concentrations indicated that S. stellata simultaneously catabolized both substrates. Furthermore, enhanced growth rates were observed when S. stellata was provided both compounds simultaneously compared to the rates of cells grown singly with an equimolar concentration of either substrate alone. This simultaneous-catabolism phenotype was also demonstrated in another lineage member, Ruegeria pomeroyi DSS-3. These findings challenge the paradigm of sequential aromatic catabolism reported for soil bacteria and contribute to the growing body of physiological evidence demonstrating the metabolic versatility of roseobacters.

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The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe(3+), Cu(2+), Zn(2+), Co(2+), Al(3+), Cd(2+), Ni(2+) and Mn(2+) ions caused 20-80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni(2+)) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.

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Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications. Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). One of the pitfalls of these systems, however, is the production of carboxylic acids. These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. Here, we present pyranose oxidase and catalase (POC) as a novel enzymatic system to perform single-molecule experiments in pH-stable conditions at arbitrary buffer strength. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. We further verify in single-molecule fluorescence experiments that POC performs as good as the common oxygen-scavenging systems, but offers long-term pH stability and more freedom in buffer conditions. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time.

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The iron(iii) complexes of the bis(phenolate) ligands 1,4-bis(2-hydroxy-4-methyl-benzyl)-1,4-diazepane H(2)(L1), 1,4-bis(2-hydroxy-4-nitrobenzyl)-1,4-diazepane H(2)(L2), 1,4-bis(2-hydroxy-3,5-dimethylbenzyl)-1,4-diazepane H(2)(L3) and 1,4-bis(2-hydroxy-3,5-di-tert-butylbenzyl)-1,4-diazepane H(2)(L4) have been isolated and studied as structural and functional models for 3,4-PCD enzymes. The complexes [Fe(L1)Cl] 1, [Fe(L2)(H(2)O)Cl] 2, [Fe(L3)Cl] 3 and [Fe(L4)Cl] 4 have been characterized using ESI-MS, elemental analysis, and absorption spectral and electrochemical methods. The single crystal X-ray structure of 3 contains the FeN(2)O(2)Cl chromophore with a novel square pyramidal (τ, 0.20) coordination geometry. The Fe-O-C bond angle (135.5°) and Fe-O bond length (1.855 Å) are very close to the Fe-O-C bond angles (133, 148°) and Fe-O(tyrosinate) bond distances (1.81, 1.91 Å) in 3,4-PCD enzyme. All the complexes exhibit two intense absorption bands in the ranges 335-383 and 493-541 nm, which are assigned respectively to phenolate (pπ) → Fe(iii) (dσ*) and phenolate (pπ) → Fe(iii) (dπ*) LMCT transitions. The Fe(iii)/Fe(ii) redox potentials of 1, 3 and 4 (E(1/2), -0.882--1.010 V) are more negative than that of 2 (E(1/2), -0.577 V) due to the presence of two electron-withdrawing p-nitrophenolate moieties in the latter enhancing the Lewis acidity of the iron(iii) center. Upon adding H(2)DBC pretreated with two equivalents of Et(3)N to the iron(iii) complexes, two catecholate-to-iron(iii) LMCT bands (656, ε, 1030; 515 nm, ε, 1330 M(-1) cm(-1)) are observed for 2; however, interestingly, an intense catecholate-to-iron(iii) LMCT band (530-541 nm) is observed for 1, 3 and 4 apart from a high intensity band in the range 451-462 nm. The adducts [Fe(L)(DBC)](-) generated from 1-4in situ in DMF/Et(3)N solution react with dioxygen to afford almost exclusively the simple two-electron oxidation product 3,5-di-tert-butylbenzoquinone (DBQ), which is discerned from the appearance and increase in intensity of the electronic spectral band around 400 nm, and smaller amounts of cleavage products. Interestingly, in DMF/piperidine the amount of quinone product decreases and those of the cleavage products increase illustrating that the stronger base piperidine enhances the concentration of the catecholate adduct. The rates of both dioxygenation and quinone formation observed in DMF/Et(3)N solution vary in the order 1 > 3 > 4 < 2 suggesting that the ligand steric hindrance to molecular oxygen attack, the Lewis acidity of the iron(iii) center and the ability of the complexes to rearrange the Fe-O phenolate bonds to accommodate the catecholate substrate dictate the extent of interaction of the complexes with substrate and hence determine the rates of reactions. This is in line with the observation of DBSQ/H(2)DBC reduction wave for the adduct [Fe(L2)(DBC)](-) at a potential (E(1/2): -0.285 V) more positive than those for the adducts of 1, 3 and 4 (E(1/2): -0.522 to -0.645 V).

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