Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.

Microbial breakdown of halogenated aromatic pesticides and related compounds

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K2) were 1.62 × 10−3 sec−1 for 3-chlorocatechol and 2.38 × 10−3 sec−1 for 3-fluorocatechol. The inhibitor constants (Ki) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

Biodegradation of halogenated organic compounds.

Publisher Summary The chapter introduces TOL plasmids and discusses pathways for toluene and xylenes catabolisms, regulation of catabolic genes, and evolution of catabolic pathways. The TOL pathway is one of three metabolic routes described by which toluene can be catabolized. The TOL plasmid encodes enzymes for conversion of the aromatic hydrocarbons to the corresponding carboxylic acids as well as the meta-pathway enzymes. The structure of the promoter sequences in pseudomonads involved in degradation of toluene to central metabolites encompasses four main elements. These are upper- and meta-pathway operon promoters and promoters of the two regulatory genes xylS and xy/R . The growth of TOL strains on benzoate (“Benzoate Curing”) is described. Uses of TOL plasmid genes in construction of novel strains and vectors are also discussed.

The TOL Plasmids: Determinants of the Catabolism of Toluene and the Xylenes

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Abstract Background Safe and clean drinking water is essential for human life. Persistent, mobile and toxic (PMT) substances and/or very persistent and very mobile (vPvM) substances are an important group of substances for which additional measures to protect water resources may be needed to avoid negative environmental and human health effects. PMT/vPvM substances do not sufficiently biodegrade in the environment, they can travel long distances with water and are toxic (those that are PMT substances) to the environment and/or human health. PMT/vPvM substance research and regulation is arguably in its infancy and in order to get in control of these substances the following (non-exhaustive list of) knowledge gaps should to be addressed: environmental occurrence; the suitability of currently available analytical methods; the effectiveness and availability of treatment technologies; the ability of regional governance and industrial stewardship to contribute to safe drinking water while supporting innovation; the ways in which policies and regulations can be used most effectively to govern these substances; and, the identification of safe and sustainable alternatives. Methods The work is the outcome of the third PMT workshop, held in March 2021, that brought together diverse scientists, regulators, NGOs, and representatives from the water sector and the chemical sector, all concerned with protecting the quality of our water resources. The online workshop was attended by over 700 people. The knowledge gaps above were discussed in the presentations given and the attendees were invited to provide their opinions about knowledge gaps related to PMT/vPvM substance research and regulation. Results Strategies to closing the knowledge, technical and practical gaps to get in control of PMT/vPvM substances can be rooted in the Chemicals Strategy for Sustainability Towards a Toxic Free Environment from the European Commission, as well as recent advances in the research and industrial stewardship. Key to closing these gaps are: (i) advancing remediation and removal strategies for PMT/vPvM substances that are already in the environment, however this is not an effective long-term strategy; (ii) clear and harmonized definitions of PMT/vPvM substances across diverse European and international legislations; (iii) ensuring wider availability of analytical methods and reference standards; (iv) addressing data gaps related to persistence, mobility and toxicity of chemical substances, particularly transformation products and those within complex substance mixtures; and (v) advancing monitoring and risk assessment tools for stewardship and regulatory compliance. The two most effective ways to get in control were identified to be source control through risk governance efforts, and enhancing market incentives for alternatives to PMT/vPvM substances by using safe and sustainable by design strategies. 

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Getting in control of persistent, mobile and toxic (PMT) and very persistent and very mobile (vPvM) substances to protect water resources: strategies from diverse perspectives

Abstract Background Lately scientific and societal concern has emerged about persistent (P), mobile (M) and toxic (T) chemicals. Such chemicals, like some polyfluoroalkyl acids (PFAAs), are of concern due to their high mobility and persistence in aquatic compartments which relates to long-term biotic exposure and difficult removal from drinking water. In this study, a screening approach for identification of PMT chemicals was developed and applied to 6158 diverse chemicals. Results Chemicals are given a continuous score for P, M and T potential based on the modelled indicators (low to moderate potential is a score of 0–0.33, high potential is a score of 0.33–0.5 and very high potential a score of 0.5–1). The P score was based on the estimated aquatic environmental half life and the M score on the chemical’s organic carbon/water partition coefficient ( K oc ) using respectively the BIOWIN3 and KocWIN QSAR models of EPISuite™. The T score was based on the indicators for five human health endpoints: carcinogenicity ( c ), mutaganicity ( m ), reprotoxicity ( r ), endocrine disruption (ED) and general repeated dose systemic toxicity. Structural alerts for these endpoints taken from the OECD QSAR Toolbox™ and Toxtree™ were used as indicators of potential (human) toxicity. Chemical similarity values to Substances of Very High Concern (SVHC) with c, m and/or r properties were also included. Value functions were developed to translate the presence of alerts and similarity to the existing SVHCs to values between 0 and 1. Subsequently, all values were also aggregated to an overall PMT score, again ranging from 0 to 1. Applying the approach to chemicals from the Inventory of Existing Commercial chemical Substances, which are also REACH registered, resulted in 15% of the chemicals receiving high scores (≥ 0.33) for all three (P-, M- and T-) indicators and 4% getting very high scores (≥ 0.5) for both the P- and M-indicators. Conclusions The approach confirmed the properties of chemicals classified as SVHC due to PMT properties (e.g. 1,4-dioxane), illustrating the ability of the approach to identify PMT chemicals of concern. Water regulators, drinking water suppliers and others can use this approach to identify potential PMT/vPvM chemicals that need further investigation. 

https://enveurope.springeropen.com/counter/pdf/10.1186/s12302-023-00749-w

J Hartmann

Papers

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Getting in control of persistent, mobile and toxic (PMT) and very persistent and very mobile (vPvM) substances to protect water resources: strategies from diverse perspectives

Screening and prioritising persistent, mobile and toxic chemicals: development and application of a robust scoring system