Biological Inspirations: Iron Complexes Mimicking the Catechol Dioxygenases

Within the broad group of Fe non-heme oxidases, our attention was focused on the catechol 1,2- and 2,3-dioxygenases, which catalyze the oxidative cleavage of aromatic rings. A large group of Fe complexes with N/O ligands, ranging from N3 to N2O2S, was developed to mimic the activity of these enzymes. The Fe complexes discussed in this work can mimic the intradiol/extradiol catechol dioxygenase reaction mechanism. Electronic effects of the substituents in the ligand affect the Lewis acidity of the Fe center, increasing the ability to activate dioxygen and enhancing the catalytic activity of the discussed biomimetic complexes. The ligand architecture, the geometric isomers of the complexes, and the substituent steric effects significantly affect the ability to bind the substrate in a monodentate and bidentate manner. The substrate binding mode determines the preferred mechanism and, consequently, the main conversion products. The preferred mechanism of action can also be affected by the solvents and their ability to form the stable complexes with the Fe center. The electrostatic interactions of micellar media, similar to SDS, also control the intradiol/extradiol mechanisms of the catechol conversion by discussed biomimetics.

Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol Degradation Haloalkaliphilic Bacterial Isolates

Haloaklophilic bacteria have a potential advantage as a bioremediation organism for high pH oil polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotypic features, biochemical characters, and 16S rRNA sequence comparison were used to identify the bacterial isolates including Halomonas HA1 and Marinobacter HA2. These strains showed high requirement of NaCl for growth specially HA1 strain that essentially required NaCl for its growth. The isolates are capable of degrading phenol at optimal pH values between 8 and 9 with the ability to grow in levels of pH up to 11, like what was seen in Halomonas HA1 strain. Both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta cleavage pathway while Marinobacter HA2 using the 2,3 ortho cleavage pathway indicated by universal primer sets for 1,2 and 2,3 CTD genes. Phenol degradation showed a comparable pattern between both isolates, while Marinobacter HA2 isolate can eliminate the added phenol within an incubation period of 72 h, The Halomonas HA1isolate required 96 h to degrade 84% of the same amount of phenol. The phylogenetic analysis of the amino acid sequence of 1,2 CTD (catechol dioxygenase) of Halomonas HA1showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae while 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Semi-quantitative RT PCR analysis proved the constitutive expression pattern of both dioxygenase genes.

Quantification of Naphthalene Dioxygenase (NahAC) and Catechol Dioxygenase (C23O) Catabolic Genes Produced by Phenanthrene-Degrading Pseudomonas fluorescens AH-40

Background: Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies. Objective: Detection of the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determining the variation in the concentration of both nahAC and C23O catabolic genes during 15 days of the incubation period. Methods: In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture were achieved by quantitative PCR. Results: Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of phenanthrene degradation. Conclusion: nahAc and C23O genes are the main marker genes for phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.

Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

Structures of an extradiol catechol dioxygenase – C23O64, from 3-nitrotoluene degrading Diaphorobacter sp. strain DS2 in substrate-free, substrate-bound and substrate analog-bound states

This manuscript reports structure–function studies of Catechol 2,3-dioxygenase (C23O64), which is the second enzyme in the metabolic degradation pathway of 3-nitrotoluene by Diaphorobacter sp. strain DS2. The recombinant protein is a ring cleavage enzyme for 3-methylcatechol and 4-methylcatechol products formed after dioxygenation of the aromatic ring. Here we report the substrate-free, substrate-bound, and substrate-analog bound crystal structures of C23O64. The protein crystallizes in the P6(2)22 space-group. The structures were determined by molecular replacement and refined to resolutions of 2.4, 2.4, 2.2 Å, respectively. A comparison of the structures with related extradiol dioxygenases showed 22 conserved residues. A comparison of the active site pocket with catechol 2,3-dioxygenase (LapB) from Pseudomonas sp KL28 and homoprotocatechuate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum shows significant similarities to suggest that the mechanism of enzyme action is similar to HPCD.

Catechol-2,3-Dioxygenase and Crude Oil Biodegradation Screening of Rhizo-Bacterial Endophytes from Bodo-Gokana, Rivers State

Strain-selection for the biotechnological application is critical in modern environmental bioremediation process design. In this study, twenty-one rhizobacterial isolates were obtained from the rhizosphere soil of Cyperus sp., Cyperus rotundus, Mariscus alternifolius and Maricus ligularis. Samples were treated using Bushnell-Haas media fortified with Bonny light crude oil plus 1% (v/v) rhizosphere soil from pre-impacted locations in Bodo-Ogoni, Gokana LGA, Rivers state. They were screened and four bacterial isolates were selected on the basis of -2,3 catechol dioxygenase activity and their growth dynamics using the growth function model in XLSTAT v 2019.1.3. Vapour-phase transfer and viable plate count techniques were employed in the determination of microbial dynamics. The order for relative enzyme activity and degradation rates followed Pseudomonas fluorescens > Achromobacter agilis > Bacillus thuringiensis > Staphylococcus lentus. The order for growth range were 7.0-10.5 Log10CFU/ml, 6.2-10.3 Log10CFU/ml, 7.1-10.1 Log10CFU/ml and 6.4-10.2 Log10CFU/ml for Achromobacter agilis > Pseudomonas fluorescens > Bacillus thuringiensis > Staphylococcus lentus. The growth pattern of these isolates fitted into the 5th order polynomial function (y= pr1+pr2*X+pr3*X2+pr4*X3+pr5*X4+pr6*X5) with R2-values of 0.999, 0.998, 0.991,0.999 compares to Gompertz and Asymptotic functions that have the least predictability with R2- values of 0.893, 0.599, 0.869, 0.894 and 0.80, 0.545, 0.829, 0.688 for the four isolates respectively. Enzyme activity of the isolates revealed that the isolates were most active on the 6th day of the study and had a lag phase within the first few hours to a day of the study. Statistical analyses revealed a significant difference using two-way ANOVA; p< 0.001 for both enzyme activity and growth rate. The results underscore the benefits and richness of rhizobacterial flora as rich in enzymatic activity for ecosystem-recovery. Overall, the study has shown the great potential and feasibility for deploying robust biotechnology for the monitoring of environmental media involving hydrocarbon pollution in the Niger Delta.

Integrated Meta-omics Approaches To Understand the Microbiome of Spontaneous Fermentation of Traditional Chinese Pu-erh Tea

ABSTRACT The microbiome in fermentation has direct impacts on the quality of fermented foods and is of great scientific and commercial interest. Despite considerable effort to explain the microbial metabolism associated with food fermentation, the role of the microbiome in pu-erh tea fermentation remains unknown. Here, we applied integrated meta-omics approaches to characterize the microbiome in two repeated fermentations of pu-erh tea. Metabarcoding analysis of bacterial 16S rRNA genes showed a decrease in the proportion of Proteobacteria and an increase in the abundance of Firmicutes during fermentation. Metabarcoding analysis of fungal internal transcribed spacer (ITS) sequence demonstrated that Rasamsonia, Thermomyces, and Aspergillus were dominant at the intermediate stage, whereas Aspergillus was dominant at other stages in fermentation. Metaproteomics analysis assigned primary microbial metabolic activity to metabolism and identified microbial carbohydrate-active enzymes involved in the degradation of polysaccharides including cellulose, xylan, xyloglucan, pectin, starch, lignin, galactomannan, and chitin. Metabolomics and high-performance liquid chromatography analysis revealed that levels of phenolic compounds, including gallates, decreased whereas contents of gallic acid and ellagic acid significantly increased after fermentation (P < 0.05). The changes in levels of gallates and gallic acid were associated with the hydrolysis of tannase. Glycoside hydrolases, phenol 2-monooxygenase, salicylaldehyde dehydrogenase, salicylate 1-monooxygenase, catechol O-methyltransferase, catechol dioxygenase, and quercetin 2,3-dioxygenases were hypothesized to be related to oxidation, conversion, or degradation of phenolic compounds. We demonstrated microbiota in fermentation and their function in the production of enzymes related to the degradation of polysaccharides, and metabolism of phenolic compounds, resulting in changes in metabolite contents and the quality of pu-erh tea. IMPORTANCE Fermented foods play important roles in diets worldwide and account for approximately one-third of all foods and beverages consumed. To date, traditional fermentation has used spontaneous fermentation. The microbiome in fermentation has direct impacts on the quality and safety of fermented foods and contributes to the preservation of traditional methods. Here, we used an integrated meta-omics approach to study the microbiome in the fermentation of pu-erh tea, which is a well-known Chinese fermented food with a special flavor and healthful benefits. This study advanced the knowledge of microbiota, metabolites, and enzymes in the fermentation of pu-erh tea. These novel insights shed light onto the complex microbiome in pu-erh fermentation and highlight the power of integrated meta-omics approaches in understanding the microbiome in food fermentation ecosystems.

Four Aromatic Intradiol Ring Cleavage Dioxygenases from Aspergillus niger

ABSTRACT Ring cleavage dioxygenases catalyze the critical ring-opening step in the catabolism of aromatic compounds. The archetypal filamentous fungus Aspergillus niger previously has been reported to be able to utilize a range of monocyclic aromatic compounds as sole sources of carbon and energy. The genome of A. niger has been sequenced, and deduced amino acid sequences from a large number of gene models show various levels of similarity to bacterial intradiol ring cleavage dioxygenases, but no corresponding enzyme has been purified and characterized. Here, the cloning, heterologous expression, purification, and biochemical characterization of four nonheme iron(III)-containing intradiol dioxygenases (NRRL3_02644, NRRL3_04787, NRRL3_05330, and NRRL3_01405) from A. niger are reported. Purified enzymes were tested for their ability to cleave model catecholate substrates, and their apparent kinetic parameters were determined. Comparisons of kcat/Km values show that NRRL3_02644 and NRRL3_05330 are specific for hydroxyquinol (1,2,4-trihydroxybenzene), and phylogenetic analysis shows that these two enzymes are related to bacterial hydroxyquinol 1,2-dioxygenases. A high-activity catechol 1,2-dioxygenase (NRRL3_04787), which is phylogenetically related to other characterized and putative fungal catechol 1,2-dioxygenases, was also identified. The fourth enzyme (NRRL3_01405) appears to be a novel homodimeric Fe(III)-containing protocatechuate 3,4-dioxygenase that is phylogenetically distantly related to heterodimeric bacterial protocatechuate 3,4-dioxygenases. These investigations provide experimental evidence for the molecular function of these proteins and open the way to further investigations of the physiological roles for these enzymes in fungal metabolism of aromatic compounds. IMPORTANCE Aromatic ring opening using molecular oxygen is one of the critical steps in the degradation of aromatic compounds by microorganisms. While enzymes catalyzing this step have been well-studied in bacteria, their counterparts from fungi are poorly characterized despite the abundance of genes annotated as ring cleavage dioxygenases in fungal genomes. Aspergillus niger degrades a variety of aromatic compounds, and its genome harbors 5 genes encoding putative intracellular intradiol dioxygenases. The ability to predict the substrate specificities of the encoded enzymes from sequence data are limited. Here, we report the characterization of four purified intradiol ring cleavage dioxygenases from A. niger, revealing two hydroxyquinol-specific dioxygenases, a catechol dioxygenase, and a unique homodimeric protocatechuate dioxygenase. Their characteristics, as well as their phylogenetic relationships to predicted ring cleavage dioxygenases from other fungal species, provide insights into their molecular functions in aromatic compound metabolism by this fungus and other fungi.