Transient accumulation of gamma-butyrolactone during degradation of bis(4-chloro-n-butyl) ether by diethylether-grown Rhodococcus sp. strain DTB.

Rhodococcus sp. strain DTB (DSM 44534) grows aerobically on diethylether as sole source of carbon and energy. Dense cell suspension experiments showed that the induced ether-cleaving enzyme system attacks a broad range of ethers like tetrahydrofuran, phenetole and chlorinated alkylethers including Calpha-substituted alkylethers. Identification of metabolites revealed that degradation of the ethers started by an initial attack of the ether bond. Diethylether-grown cells degraded bis(4-chloro-n-butyl) ether via an initial ether scission followed by the transient accumulation of gamma-butyrolactone as intermediate at nearly stoichiometric concentrations.

Transformation of 2,2'-dichlorodiisopropyl ether in mixed and pure culture.

An aerobic enrichment culture derived from a groundwater contaminated with organic and chloroorganic compounds was adapted to the transformation of 2,2'-dichlorodiisopropyl ether (DDE) in a continuous fixed-bed bioreactor. Continuous DDE removal efficiencies over 90% were achieved with a model water containing 3.3 mM methanol as co-substrate at DDE loading rates of up to 150 micromol l(-1) day(-1) with a hydraulic retention time of 24 h. In batch cultures, a stoichiometric release of 2 micromol chloride per micromol DDE transformed was observed. From the mixed culture, a strain was isolated that is able to grow on DDE as the sole energy and carbon source, tolerating DDE concentrations of up to 1 mM. Based on 16S rRNA sequencing, the strain is affiliated with the genus Rhodococcus.

Bacterial dehalorespiration with chlorinated benzenes.

Chlorobenzenes are toxic, highly persistent and ubiquitously distributed environmental contaminants that accumulate in the food chain. The only known microbial transformation of 1,2,3,5-tetrachlorobenzene (TeCB) and higher chlorinated benzenes is the reductive dechlorination to lower chlorinated benzenes under anaerobic conditions observed with mixed bacterial cultures. The lower chlorinated benzenes can subsequently be mineralized by aerobic bacteria. Here we describe the isolation of the oxygen-sensitive strain CBDB1, a pure culture capable of reductive dechlorination of chlorobenzenes. Strain CBDB1 is a highly specialized bacterium that stoichiometrically dechlorinates 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, 1,2,3,4-TeCB, 1,2,3,5-TeCB and 1,2,4,5-TeCB to dichlorobenzenes or 1,3,5-TCB. The presence of chlorobenzene as an electron acceptor and hydrogen as an electron donor is essential for growth, and indicates that strain CBDB1 meets its energy needs by a dehalorespiratory process. According to their 16S rRNA gene sequences, strain CBDB1, Dehalococcoides ethenogenes and several uncultivated bacteria form a new bacterial cluster, of which strain CBDB1 is the first, so far, to thrive on a purely synthetic medium.

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity.

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement. Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.

Bacterial growth based on reductive dechlorination of trichlorobenzenes.

An anaerobic mixed bacterial culture was enriched for bacteria dechlorinating 1,2,3- and 1,2,4-trichlorobenzene (TCB) to dichlorobenzenes by exclusive use of non-fermentable substrates and the application of vancomycin. Growth and dechlorination occurred in a purely synthetic medium with formate or hydrogen, acetate, and TCB. Neither acetogenesis nor methanogenesis was detected in the culture. Repeated subculturing maintaining high dechlorinating activities was also achieved when only hydrogen and TCB were supplied. This indicated that reductive dechlorination of TCB was the primary energy conservating process. The number of dechlorinating bacteria was strictly limited by the amount of TCB supplied in the medium. In addition, the dechlorinating activity could be maintained only in the presence of TCB. A most probable number analysis showed that the dechlorinating species amounted to at least 6 x 10(5) cells per ml at a total cell number of about 2 x 10(6) cells per ml. Vitamin B12 significantly stimulated the dechlorinating activity.

Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer--sequence of the gene and deduced structural properties of the enzyme.

The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline-quinone-dependent periplasmic and membrane-bound dehydrogenases acting on alcohols, glucose and quinate or shikimate. It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X-ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline-quinone-dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes. Despite the sequence similarity between the large a subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60-kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large alpha subunit, the expression of a small beta subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217-227].

Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene.

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1.

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1, 4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1, 3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates.

Purification and characterization of chlorobenzene cis-dihydrodiol dehydrogenase from Xanthobacter flavus 14p1.

Chlorobenzene cis-dihydrodiol dehydrogenase was purified to homogeneity from Xanthobacter flavus 14p1, which used 1,4-dichlorobenzene as the sole source of carbon and energy. The enzyme converted a number of halogenated substrates with high specific activity. The pI of the native chlorobenzene cis-dihydrodiol dehydrogenase was 5.4, and the molecular mass was approximately 100 kDa, as determined by gel filtration. The enzyme was composed of four apparently identical subunits with a molecular mass of 26.5 kDa. The Michaelis constant for 3,6-dichlorobenzene cis-dihydrodiol (210 microM) was lower than for benzene cis-dihydrodiol (780 microM), while the specific activity with benzene cis-dihydrodiol (63 units/ mg) was higher than with 3,6-dichlorobenzene cis-dihydrodiol (32 units/mg). Chlorobenzene cis-dihydrodiol dehydrogenase accepted also NADP+ as cosubstrate; however, the activity was reduced to 14% of that with NAD+. The enzymic activity was inhibited by mercuric chloride and to a lesser extent by the metal-ion chelators 8-hydroxyquinoline and KCN.

Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1.

Xanthobacter flavus 14p1 was isolated from sludge of the river Mulde by selective enrichment with 1,4-dichlorobenzene as the sole source of carbon and energy. The bacterium did not use other aromatic or chloroaromatic compounds as growth substrates. During growth on 1,4-dichlorobenzene, stoichiometric amounts of chloride ions were released. Degradation products of 1,4-dichlorobenzene were identified by gas chromatography-mass spectrometry analysis. 3,6-Dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene and 3,6-dichlorocatechol were isolated from culture fluid. 2,5-Dichloromuconic acid and 2-chloromaleylacetic acid as well as the decarboxylation product 2-chloroacetoacrylic acid were identified after enzymatic conversion of 3,6-dichlorocatechol by cell extract. 1,4-Dichlorobenzene dioxygenase, dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase activity were induced in cells grown on 1,4-dichlorobenzene. The results demonstrate that 1,4-dichlorobenzene degradation is initiated by dioxygenation and that ring opening proceeds via ortho cleavage.

Cytochrome c550 from Pseudomonas aeruginosa.

In cells of Pseudomonas aeruginosa A.T.C.C. 17933 grown on ethanol the synthesis of a soluble c-type cytochrome, together with quinoprotein ethanol dehydrogenase, is induced. The cytochrome, with an alpha-absorption band at 550 nm, was purified to homogeneity. The molecular mass of the monomeric protein is 15 kDa, the pI is 4.8, and it contains one haem prosthetic group. The midpoint potential of the autoxidizable, but not autoreducible, cytochrome is 280 mV. Cytochrome c550 mediates electron transfer between quinoprotein ethanol dehydrogenase and ferricyanide. In a system composed of membrane particles with NN'NN'-tetramethyl-p-phenylenediamine oxidase activity and quinoprotein ethanol dehydrogenase, oxygen consumption is only observed in the presence of cytochrome c550. This indicates the participation of the cytochrome in the electron-transport chain linked to quinoprotein ethanol dehydrogenase in P. aeruginosa. The electron transport from ethanol dehydrogenase to oxygen is inhibited by myxothiazol and antimycin, indicating that a cytochrome bc1-like complex is involved.

A highly specific D-hydroxyisovalerate dehydrogenase from the enniatin producer Fusarium sambucinum.

A highly specific D-hydroxyisovalerate (D-HIV) dehydrogenase, which is a key enzyme in depsipeptide synthesis, was purified to near homogeneity from the enniatin-producing fungus Fusarium sambucinum. The enzyme catalyzes the reversible reaction of 2-ketoisovalerate (2-KIV) to D-HIV. It is strictly dependent on NADPH and exhibits a high substrate specificity with respect to 2-KIV. NADH was not accepted by the enzyme. Km values for 2-KIV and NADPH were found to be 200 and 333 microM, respectively. D-HIV dehydrogenase consists of a single polypeptide chain with a molecular mass of about 53 kDa. Optimum temperature for the reduction of 2-KIV was 35 degrees C and for the oxidation reaction was 45 degrees C. The optimum pH was found to be 7 for the reduction and 8-9 for the oxidation reaction.

Preliminary X-ray crystallographic study of malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius.

Malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius have been crystallized and characterized by X-ray diffraction measurements. Crystals of the enzyme from T. acidophilum display space-group symmetry P2(1), a = 63 A, b = 135 A, c = 83 A and beta = 105 degrees; they scattered to approximately 4 A resolution. Two crystal modifications of malate dehydrogenase from S. acidocaldarius were characterized; one displayed trigonal symmetry corresponding to space groups P321, P3(1)21 or P3(2)21 with lattice parameters a = 151 A and c = 248 A and with resolution approximately to 5 A, whereas the other modification displayed space group symmetry I23 or I2(1)3 with lattice parameters a = 129 A and approximately 4.5 A resolution.

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported.

Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation.

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.

Quinoprotein ethanol dehydrogenase from Pseudomonas.

Dye-linked ethanol dehydrogenases from Pseudomonas aeruginosa ATCC 17,933 and P. putida ATCC 17,421 were purified to homogeneity and crystallized. The amino acid composition of the two enzymes is very similar and the number of the aromatic amino acid residues found per subunit are almost identical. With respect to their catalytic and molecular properties both ethanol dehydrogenases are similar to the quinoprotein methanol dehydrogenases known from methylotrophic bacteria. They show a high pH-optimum, need ammonia or an amine as activator and are dimers of identical subunits of a molecular mass of 60,000. The dimer is the catalytically active form. Each subunit carries one prosthetic group pyrroloquinoline quinone, which can be titrated by the suicide substrate cyclopropanone ethylhemiketal. In contrast to the general methanol dehydrogenases the two ethanol dehydrogenases have a low affinity for methanol and in addition to primary alcohols they also oxidize secondary alcohols. With secondary alcohols preferentially one of the two enantiomers is oxidized. The catalytic and spectral properties of the two enzymes are very similar to the quinoprotein ethanol dehydrogenase isolated from P. aeruginosa LMD 80.53 (Groen et al., 1984. Biochem. J. 223: 921-924). However this enzyme is reported to be a monomer of molecular mass 100,000.

Archaebacterial malate dehydrogenase: the amino-terminal sequence of the enzyme from Sulfolobus acidocaldarius is homologous to the eubacterial and eukaryotic malate dehydrogenases.

42 residues of the N-terminal amino acid sequence of malate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been determined as VKVAFIGVGRGVGQTIAYNTIVNGYADEVMLYDVVPELPTKK. In eubacterial and eukaryotic enzymes this region is known to encompass residues involved in pyridine nucleotide binding. In the archaebacterial enzyme the residues Gly-7, Gly-11 and Asp-33 are also present. The data suggest that in the enzyme from S. acidocaldarius like in the other malate dehydrogenases the binding domain for NAD(H) is localized at the N-terminal part of the polypeptide chain. The archaebacterial enzyme is homologous to the other malate dehydrogenases, of which the amino acid sequences are known, however, it is only distantly related to the mitochondrial/E. coli group and the cytosolic/Thermus flavus group.

Drop dialysis: time course of salt and protein exchange.

By drop dialysis with membrane filters of 25 or 50 nm average pore size, salt concentrations are reduced to 15% within 25 min. During this time only 10% of ribonuclease with a Mr 13,500 will diffuse in and through the membrane. However, in the presence of 1 M NaCl about 25% of the enzyme is lost. The difference in the rate of salt removal and enzyme loss is caused by the difference in diffusion constants. Therefore with enzymes of higher molecular weights, less protein will be lost, as is shown with beta-galactose dehydrogenase. This enzyme with Mr 64,000 is lost at a lower rate than ribonuclease. The net charge of a protein apparently does not influence the rate with which it diffuses through the membrane. The time course of salt and protein exchange was studied to provide data for estimating the optimal conditions for the required reduction in salt concentration. To prepare small protein samples for electrophoresis or other analytical methods, which require low salt concentrations or a buffer change, drop dialysis is a fast and effective method with tolerable loss of protein.

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.