Detoxification of reactive intermediates during microbial metabolism of halogenated compounds.

The reactivity and toxicity of metabolic intermediates that are generated by initial biotransformation reactions can be a major limiting factor for biodegradation of halogenated organic compounds. Recent work on the conversion of haloalkanes, chloroaromatics and chloroethenes indicates that microorganisms may become less sensitive to toxic effects either by using novel pathways that circumvent the generation of reactive intermediates or by producing modified enzymes that decrease the toxicity of such compounds.

Degradation of halogenated aliphatic compounds: the role of adaptation.

A limited number of halogenated aliphatic compounds can serve as a growth substrate for aerobic microorganisms. Such cultures have (specifically) developed a variety of enzyme systems to degrade these compounds. Dehalogenations are of critical importance. Various heavily chlorinated compounds are not easily biodegraded, although there are no obvious biochemical or thermodynamic reasons why microorganisms should not be able to grow with any halogenated compound. The very diversity of catabolic enzymes present in cultures that degrade halogenated aliphatics and the occurrence of molecular mechanisms for genetic adaptation serve as good starting points for the evolution of catabolic pathways for compounds that are currently still resistant to biodegradation.

Allantoinase from Pseudomonas aeruginosa. Purification, properties and immunochemical characterization of its in vivo inactivation.

The catabolic enzyme allantoinase is rapidly inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. This process is irreversible since the protein synthesis inhibitor chloramphenicol completely blocked the reappearance of allantoinase activity that is observed when allantoin is added to stationary cells. Purified alloantoinase appeared to be a protein composed of four identical subunits with a molecular weight of 38,000. With antibodies raised against purified allantoinase it was found that allantoinase inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that allantoinase inactivation is caused or followed by rapid proteolysis.

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.

Engineering proteins for environmental applications.

Recently, significant new insight has been obtained into the structure and catalytic mechanism of enzymes that convert environmental pollutants. Recent advances in protein engineering make it possible to use this information for improving the catalytic performance of such enzymes to achieve increased stability and expanded substrate range.

Microbial dehalogenation.

Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive dehalogenases are extra-cytoplasmic corrinoid-containing iron-sulphur proteins. Sequence analysis and mutagenesis studies indicate that several dehalogenases are homologous to enzymes that carry out transformations on non-halogenated substrates.

Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes.

Haloalkane dehalogenase (DhlA) hydrolyzes short-chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three-step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three-step route involves different and perhaps larger conformational changes than the two-step export route. We propose that the three-step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two-step route for halide release involves the transfer of the halide ion from the halide-binding site in the cavity to a binding site somewhere at the protein surface, where a so-called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements are necessary for this latter process.

Crystallization and preliminary X-ray analysis of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10.

Haloacid dehalogenases are enzymes that cleave carbon-chlorine or carbon-bromine bonds of 2-haloalkanoates. X-ray-quality crystals of L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8-7.0, using macroseeding techniques. The crystals, which diffract in the X-ray beam up to 2.0 A resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 A, b = 93.1 A, c = 84.2 A. A native data set to 2.3 A has been collected, with a completeness of 97% and an Rsym of 6.0%.

Activation of an Asp-124-->Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine.

Haloalkane dehalogenase hydrolyses various 1-halo-n-alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl-enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp-124) that displaces the halogen during formation of the intermediate was changed to an amide by site-directed mutagenesis (Asp-124-->Asn). Activity measurements and analysis of peptides containing the nucleophilic residue showed that the mutant enzyme was inactive, but that the activity increased by rapid deamidation of the asparagine residue, yielding wild type enzyme. There was no indication for isoaspartate formation during this process. The results suggest that a water molecule that is located close to the carboxyl function of Asp-124 in the X-ray structure is highly reactive and is responsible for the observed deamidation.

Genetic adaptation of bacteria to halogenated aliphatic compounds.

The bacterial degradation and detoxification of chlorinated xenobiotic compounds requires the production of enzymes that are capable of recognizing and converting compounds which do not occur at significant concentrations in nature. We have studied the catabolic route of 1,2-dichloroethane as an example of a pathway for the conversion of such a synthetic compound. In strains of Xanthobacter and Ancylobacter that have been isolated on 1,2-dichloroethane, the first catabolic step is catalyzed by a hydrolytic haloalkane dehalogenase. The enzyme converts 1,2-dichloroethane to 2-chloroethanol but is also active with many other environmentally important haloalkanes such as methylchloride, methylbromide, 1,2-dibromoethane, epichlorohydrin, and 1,3-dichloropropene. Further degradation of 2-chloroethanol proceeds by oxidation to the carboxylic acid and dehalogenation to glycolate. The aldehyde dehydrogenase prevents toxicity of the reactive chloroacetaldehyde that is formed as an intermediate and is necessary for establishing a functional 2-chloroethanol degradative pathway in a strain that is not capable of growth on this compound.

Highly enantioselective and regioselective biocatalytic azidolysis of aromatic epoxides.

[figure: see text] The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides. By means of kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee. In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.

Purification and characterization of a surface-binding protein from Lactobacillus fermentum RC-14 that inhibits adhesion of Enterococcus faecalis 1131.

Lactobacilli have been shown to be important in the maintenance of the healthy urogenital flora. One strain, Lactobacillus fermentum RC-14, releases surface-active components which can inhibit adhesion of uropathogenic bacteria. Using a quantitative method for determining inhibition of adhesion, a protein with high anti-adhesive properties against Enterococcus faecalis 1131 was purified. The N-terminal sequence of the 29-kDa protein was identical to that of a collagen-binding protein from Lactobacillus reuteri NCIB 11951, and exhibited close homology with a basic surface protein from L. fermentum BR11. The results suggest that this anti-adhesive cell surface protein of Lactobacillus could protect against uropathogens by preventing their adhesion. the Federation of European Microbiological Societies.

Formation and detoxification of reactive intermediates in the metabolism of chlorinated ethenes.

Short-chain halogenated aliphatics, such as chlorinated ethenes, constitute a large group of priority pollutants. This paper gives an overview on the chemical and physical properties of chlorinated aliphatics that are critical in determining their toxicological characteristics and recalcitrance to biodegradation. The toxic effects and principle metabolic pathways of halogenated ethenes in mammals are briefly discussed. Furthermore, the bacterial degradation of halogenated compounds is reviewed and it is described how product toxicity may explain why most chlorinated ethenes are only degraded cometabolically under aerobic conditions. The cometabolic degradation of chlorinated ethenes by oxygenase-producing microorganisms has been extensively studied. The physiology and bioremediation potential of methanotrophs has been well characterized and an overview of the available data on these organisms is presented. The sensitivity of methanotrophs to product toxicity is a major limitation for the transformation of chlorinated ethenes by these organisms. Most toxic effects arise from the inability to detoxify the reactive chlorinated epoxyethanes occurring as primary metabolites. Therefore, the last part of this review focuses on the metabolic reactions and enzymes that are involved in the detoxification of epoxides in mammals. A key role is played by glutathione S-transferases. Furthermore, an overview is presented on the current knowledge about bacterial enzymes involved in the metabolism of epoxides. Such enzymes might be useful for detoxifying chlorinated ethene epoxides and an example of a glutathione S-transferase with activity for dichloroepoxyethane is highlighted.

NADH-Regulated metabolic model for growth of Methylosinus trichosporiumOB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance.

A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis-Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth-based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash-out of suspended cultures at low TCE loads (below microM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash-out. Pulsed addition of TCE (0.014-0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33-0.64 mM). This emphasizes the inefficient use of enzyme activity in growth-based systems. A comparison of growth-based and similar non-growth-based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two-step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.

NADH-Regulated metabolic model for growth of Methylosinus trichosporium OB3b. Model presentation, parameter estimation, and model validation.

A biochemical model is presented that describes growth of Methylosinus trichosporium OB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly-beta-hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor-instead of the commonly applied energy carrier ATP. This way a stable and well-regulated growth model is obtained that gives a realistic description of a variety of steady-state and transient-state experimental data. An analysis of the cells' physiological properties is given to illustrate the applicability of the model. Steady-state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in V(MAX) values is necessary to describe growth at different dilution rates, the organism seems to have a "rigid enzyme system", the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power.

Developments in biotechnology of relevance to drinking water preparation.

This paper discusses strategies to increase the feasibility of microorganisms for the removal of toxic xenobiotics from waste water and drinking water. Based on the principles of adaptational mutations and genetic exchange of catabolic activities, it becomes possible to select and engineer microorganisms that are suitable for the degradation of recalcitrant compounds. The detailed biochemical knowledge that is required for this is now rapidly evolving, and especially for the degradation of chlorinated organics several detoxifying dehalogenation mechanisms have been studied in detail. The feasibility of specialized bacteria for waste and water treatment will be dependent on the possibility to obtain stable performance and maintenance in treatment systems.

Mutation of Residue ßF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties.

Residue phenylalanine 71 of the ß-chain of penicillin acylase from E. coli is involved in substrate binding and chiral discrimination of its enantiomers. Different amino acid residues have been introduced at position ßF71, and the mutants were studied with respect to their enantioselectivity and substrate specificity. Some mutants demonstrated remarkably improved catalytic activity. Moreover, mutation of ßF71 residue allowed to enhance penicillin acylase enantioselectivity. The catalytic activity to the specific substrates was improved up to 36 times, most notably for K, R, and L mutants. Increased activity to a D-phenylglycine derivative - a valuable specificity improvement for biocatalytic synthesis of new penicillins and cephalosporins - was shown for ßF71R and ßF71L mutants. The synthetic capacity of penicillin acylase with 6-aminopenicillanic acid as an external nucleophile was especially sensitive to mutation of the ß71 residue in contrast to the synthesis with 7-aminodeacetoxycephalosporanic acid.

Enantioselective formation and ring-opening of epoxides catalysed by halohydrin dehalogenases.

Halohydrin dehalogenases catalyse the conversion of vicinal halohydrins into their corresponding epoxides, while releasing halide ions. They can be found in several bacteria that use halogenated alcohols or compounds that are degraded via halohydrins as a carbon source for growth. Biochemical and structural studies have shown that halohydrin dehalogenases are evolutionarily and mechanistically related to enzymes of the SDR (short-chain dehydrogenase/reductase) superfamily. In the reverse reaction, which is epoxide-ring opening, different nucleophiles can be accepted, including azide, nitrite and cyanide. This remarkable catalytic promiscuity allows the enzymatic production of a broad range of beta-substituted alcohols from epoxides. In these oxirane-ring-opening reactions, the halohydrin dehalogenase from Agrobacterium radiobacter displays high enantioselectivity, making it possible to use the enzyme for the preparation of enantiopure building blocks for fine chemicals.

Kinetics of halide release of haloalkane dehalogenase: evidence for a slow conformational change.

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides. The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity, but the kcat values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s-1, respectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall kcat. Furthermore, at 5mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s-1 (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5mM 1,2-dibromoethane occurred at a rate of only 8 s-1. The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of kobs on the halide concentration suggested that there were two parallel routes for halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations. The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s-1 for bromide and chloride, respectively). We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion. A solvent kinetic isotope effect of 2H2O was found both on kcat and on the rate of halide release. 2H2O mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of 2H2O on the conformation of proteins.

Kinetic mechanism of the enantioselective conversion of styrene oxide by epoxide hydrolase from Agrobacterium radiobacter AD1.

Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16. The (R)-enantiomer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1). The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques. For (R)-styrene oxide a four-step mechanism was needed to describe the data. It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step. The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established [Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. Soc. 118, 9436-9437]. For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant K(S) due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps. The combined reaction schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide.