Strain CBS3 Research Articles

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Investigation of the Catalytic Mechanism of the Hotdog-Fold Enzyme Superfamily Pseudomonas sp. Strain CBS3 4-Hydroxybenzoyl-CoA Thioesterase

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.

Isolation and characterization of monochloroacetic acid-degrading bacteria

Five Burkholderia strains (CL-1, CL-2, CL-3, CL-4, and CL-5) capable of degrading monochloroacetic acid (MCA) were isolated from activated sludge or soil samples gathered from several parts of Japan. All five isolates were able to grow on MCA as the sole source of carbon and energy, and argentometry and gas chromatography-mass spectroscopy analyses showed that these five strains consumed MCA completely and released chloride ions stoichiometrically within 25 h. The five isolates also grew on monobromoacetic acid, monoiodoacetic acid, and L-2-monochloropropionic acid as sole sources of carbon and energy. In addition, the five isolates could not grow with DCA but dehalogenate single chlorine from DCA. Because PCR analyses revealed that all five isolates have an identical group II dehalogenase gene fragment and no group I deh gene, only strain CL-1 was analyzed further. The partial amino acid sequence of the group II dehalogenase of strain CL-1, named DehCL1, showed 74.6% and 65.2% identities to corresponding regions of the two MCA dehalogenases, DehCI from Pseudomonas sp. strain CBS-3 and Hdl IVa from Burkholderia cepacia strain MBA4, respectively. The secondary-structure motifs of the haloacid dehalogenase (HAD) superfamily and the amino acid residues involved in substrate binding, catalysis, and hydrophobic pocket formation were conserved in the partial amino acid sequence of DehCL1.

The Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. strain SU

The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel beta-sheet flanked on one side by a rather long alpha-helix. The dimers come together to form the tetramer with their alpha-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their alpha-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.

Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site.

4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (Thoden, J. B., Holden, H. M., Zhuang, Z. and Dunaway-Mariano, D. (2002) X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase, J. Biol. Chem., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.

X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase

The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase. The structure of the unbound form of this thioesterase has been shown to contain the so-called "hot dog" fold with a large helix packed against a five-stranded anti-parallel beta-sheet. To address the manner in which the enzyme accommodates the substrate within the active site, two inhibitors have been synthesized, namely 4-hydroxyphenacyl-CoA and 4-hydroxybenzyl-CoA. Here we describe the structural analyses of the enzyme complexed with these two inhibitors determined and refined to 1.5 and 1.8 A resolution, respectively. These studies indicate that only one protein side chain, Ser(91), participates directly in ligand binding. All of the other interactions between the protein and the inhibitors are mediated through backbone peptidic NH groups, carbonyl oxygens, and/or solvents. The structures of the enzyme-inhibitor complexes suggest that both a hydrogen bond and the positive end of a helix dipole moment serve to polarize the electrons away from the carbonyl carbon of the acyl group, thereby making it more susceptible to nucleophilic attack. Additionally, these studies demonstrate that the carboxylate group of Asp(17) is approximately 3.2 A from the carbonyl carbon of the acyl group. To address the role of Asp(17), the structure of the site-directed mutant protein D17N with bound substrate has also been determined. Taken together, these investigations suggest that the reaction mechanism may proceed through an acyl enzyme intermediate.

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl- N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.

Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4.

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.

The use of quantitative PCR, plant and earthworm bioassays, plating and chemical analysis to monitor 4-chlorobiphenyl biodegradation in soil microcosms

4-Chlorobiphenyl biodegradation was monitored using several methods including molecular detection, microbiological and ecotoxicological techniques and analytical tools. Experiments were carried out in microcosms with artificial soil and an uncultivated sandy loam soil, contaminated at a rate of 1 g kg −1 of 4-chlorobiphenyl. Two strains were used for bioremediation: a 4-chlorobiphenyl degrader, the bacterial strain Pseudomonas sp. strain B4 and the bacterial strain Pseudomonas sp. strain CBS3 which is able to mineralize 4-chlorobenzoate. Molecular tools were constructed for the detection and the quantification of dihydroxybiphenyl dioxygenase gene ( bphC) from Pseudomonas sp. strain B4 and for the detection of the 4-chlorobenzoate coenzyme A dehalogenase gene from Pseudomonas sp. strain CBS3. Evidence was found for 4-chlorobiphenyl toxicity to barley and earthworms using standardized ecotoxicological tests. When Pseudomonas sp. strain B4 was introduced alone into microcosms, no decrease in toxicity occurred although 4-chlorobiphenyl was entirely degraded. The introduction of Pseudomonas sp. strain CBS3 into inoculated microcosms resulted in the disappearance of the 4-chlorobenzoate accumulated during the degradation of 4-chlorobiphenyl by Pseudomonas sp. strain B4, and in the disappearance of any toxic effect indicating that remediation was complete. Similar results were obtained independent of the nature of the soil used in microcosms. These results underline the need for taking biological effects into account in order to assess remediation efficiency.

The Three-dimensional Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Pseudomonas sp. Strain CBS-3

The soil-dwelling microbe, Pseudomonas sp. strain CBS-3, has attracted recent attention due to its ability to survive on 4-chlorobenzoate as its sole carbon source. The biochemical pathway by which this organism converts 4-chlorobenzoate to 4-hydroxybenzoate consists of three enzymes: 4-chlorobenzoyl-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl-CoA thioesterase. Here we describe the three-dimensional structure of the thioesterase determined to 2.0-A resolution. Each subunit of the homotetramer is characterized by a five-stranded anti-parallel beta-sheet and three major alpha-helices. While previous amino acid sequence analyses failed to reveal any similarity between this thioesterase and other known proteins, the results from this study clearly demonstrate that the molecular architecture of 4-hydroxybenzoyl-CoA thioesterase is topologically equivalent to that observed for beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli. On the basis of the structural similarity between these two enzymes, the active site of the thioesterase has been identified and a catalytic mechanism proposed.

Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase.

4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis.

4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction. The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme. 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1. The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1. Single-turnover reactions of [14C]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1. The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an enzyme catalyst generated via adaptive mutation.

Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3. This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging. Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 A resolution. The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer. Each subunit of the trimer folds into two distinct motifs. The larger, N-terminal domain is characterized by 10 strands of beta-pleated sheet that form two distinct layers which lie nearly perpendicular to one another. These layers of beta-sheet are flanked on either side by alpha-helices. The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic alpha-helices. This smaller domain is primarily involved in trimerization. The two domains of the subunit are linked together by a cation, most likely a calcium ion. The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed. From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step. Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase.

On the origins and functions of the enzymes of the 4-chlorobenzoate to 4-hydroxybenzoate converting pathway

This review examines the enzymes of 4-chlorobenzoate to 4-hydroxybenzoate converting pathway found in certain soil bacteria. This pathway consists of three enzymes: 4-chlorobenzoate: Coenzyme A ligase, 4-chlorobenzoyl-Coenzyme A dehalogenase and 4-hydroxybenzoyl-Coenzyme A thioesterase. Recent progress made in the cloning and expression of the pathway genes from assorted bacterial strains is described. Gene order and sequence found among these strains are compared to reveal independent enzyme recruitment strategies. Sequence alignments made between the Pseudomonas sp. strain CBS3 4-chlorobenzoate pathway enzymes and structurally related proteins contained within the protein sequence data banks suggest possible origins in preexisting beta-oxidation pathways. The purification and characterization of the physical and kinetic properties of the pathway enzymes are described. Where possible a comparison of these properties between like enzymes from different bacterial sources are made.

Purification and characterization of 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain 4-CB1.

4-Chlorobenzoyl coenzyme A dehalogenase was purified to homogeneity from Arthrobacter sp. strain 4-CB1 (previously known as Acinetobacter sp. strain 4-CB1), a bacterium isolated from PCB-contaminated soil. Purification was accomplished by four chromatographic steps, including a novel affinity chromatography step. 4-Chlorobenzoyl CoA dehalogenase is a homotetramer of 33-kDa subunits with an isoelectric point of 6.1. The enzyme is stable between pH 6.5 and 10. The optimum pH for kcat is pH 8. The enzyme is able to dehalogenate substrates bearing fluorine, chlorine, bromine and iodine in the 4-position, although the rate of dehalogenation of 4-fluorobenzoyl CoA is quite slow. The enzyme is specific for dehalogenation at the 4-position, as 3-chloro- and 2-chlorobenzoyl CoA are not dehalogenated. The N-terminal sequence of the Arthrobacter sp. strain 4-CB1 dehalogenase is almost identical to that of the 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain SU and shows 30% identity to that from Pseudomonas sp. strain CBS-3.

Metabolism of both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.

Site-directed mutagenesis of the 2-haloalkanoic acid dehalogenase I gene from Pseudomonas sp. strain CBS3 and its effect on catalytic activity.

Two 2-haloalkanoic acid dehalogenases from Pseudomonas sp. strain CBS3 catalyse hydrolytic dehalogenation of chloroacetate and 2-chloropropionate. We used site-directed mutagenesis to introduce specific changes in the dehalogenase I encoding gene (dehCI). Substitution of Asp-10 by Ala-10 resulted in complete loss of dehalogenating activity although expression of the 2-haloacid dehalogenase I was not affected in the mutant as shown by western blot analysis, and although comparison of the mutated enzyme with the wild type enzyme indicated that extensive rearrangements in the three-dimensional structure of the enzyme had not occurred. From these data we suggest that Asp-10 of 2-haloacid dehalogenases I from Pseudomonas sp. strain CBS3 may be the nucleophilic residue in the active-site of this enzyme essential for halide release.

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.

Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp.-CBS3 in Escherichia coli and identification of the gene translation products.

The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli-pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed.

Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS-3.

The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C. Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA. The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A. Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.