Kegg Pathway: 3-Chloroacrylic acid degradation

Biochemistry. 2007 Aug 21; 46(33): 9596-604
Poelarends GJ, Johnson WH, Serrano H, Whitman CP

The enzymatic conversion of cis- or trans-3-Chloroacrylic acid to malonate semialdehyde is a key step in the bacterial degradation of the nematocide 1,3-dichloropropene. Two mechanisms have been proposed for the isomer-specific hydrolytic dehalogenases, cis- and trans-3-Chloroacrylic acid dehalogenase (cis-CaaD and CaaD, respectively), responsible for this step. In one mechanism, the enol isomer of malonate semialdehyde is produced by the alpha,beta-elimination of HCl from an initial halohydrin species. Phenylenolpyruvate has now been found to be a substrate for CaaD with a kcat/Km value that approaches the one determined for the CaaD reaction using trans-3-chloroacrylate. Moreover, the reaction is stereoselective, generating the 3S isomer of [3-2H]phenylpyruvate in a 1.8:1 ratio in 2H2O. These two observations and a kinetic analysis of active site mutants of CaaD suggest that the active site of CaaD is responsible for the phenylpyruvate tautomerase (PPT) activity. The activity is a striking example of catalytic promiscuity and could reflect the presence of an enol intermediate in CaaD-mediated dehalogenation of trans-3-chloroacrylate. CaaD and cis-CaaD represent different families in the tautomerase superfamily, a group of structurally homologous proteins characterized by a core beta-alpha-beta building block and a catalytic Pro-1. The eukaryotic immunoregulatory protein known as macrophage migration inhibitory factor (MIF), also a tautomerase superfamily member, exhibits a PPT activity, but the biological relevance is unknown. In addition to the mechanistic implications, these results establish a functional link between CaaD and the superfamily tautomerases, highlight the catalytic and binding promiscuity of the beta-alpha-beta scaffold, and suggest that the PPT activity of MIF could reflect a partial reaction in an unknown MIF-catalyzed reaction.

J Biol Chem. 2007 Jan 26; 282(4): 2440-9
de Jong RM, Bazzacco P, Poelarends GJ, Johnson WH, Kim YJ, Burks EA, Serrano H, Thunnissen AM, Whitman CP, Dijkstra BW

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-Chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.

Biochemistry. 2006 Jun 27; 45(25): 7700-8
Poelarends GJ, Almrud JJ, Serrano H, Darty JE, Johnson WH, Hackert ML, Whitman CP

4-Oxalocrotonate tautomerase (4-OT) and trans-3-Chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

Biochemistry. 2005 Nov 15; 44(45): 14818-27
Almrud JJ, Poelarends GJ, Johnson WH, Serrano H, Hackert ML, Whitman CP

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-Chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.

Proc Natl Acad Sci U S A. 2005 Nov 8; 102(45): 16199-202
Horvat CM, Wolfenden RV

The soil of potato fields in The Netherlands harbors bacteria with the ability to metabolize 3-Chloroacrylic acid, generated by the degradation of a pesticide (1,3-dichloropropene) that entered the environment in 1946. From examination of rate constants at elevated temperatures, we infer that the half-time at 25 degrees C for spontaneous hydrolytic dechlorination of trans-3-Chloroacrylic acid is 10,000 years, several orders of magnitude longer than half-times for spontaneous decomposition of other environmental pollutants such as 1,2-dichloroethane (72 years), paraoxon (13 months), atrazine (5 months), and aziridine (52 h). With thermodynamic parameters for activation similar to those for the spontaneous hydration of fumarate at pH 6.8, this slow reaction proceeds at a constant rate through the pH range between 2 and 12. However, at the active site of the enzyme 3-chloroacrylate dehalogenase (CaaD), isolated from a pseudomonad growing in these soils, hydrolytic dechlorination proceeds with a half-time of 0.18 s. Neither k(cat) nor k(cat)/K(m) is reduced by increasing solvent viscosity with trehalose, implying that the rate of enzymatic dechlorination is controlled by chemical events in catalysis rather than by diffusion-limited substrate binding or product release. CaaD achieves an approximately 10(12)-fold rate enhancement, matching or surpassing the rate enhancements produced by many enzymes that act on more conventional biological substrates. One of those enzymes is 4-oxalocrotonate tautomerase, with which CaaD seems to share a common evolutionary origin.

Biochemistry. 2005 Jul 5; 44(26): 9375-81
Poelarends GJ, Serrano H, Johnson WH, Whitman CP

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-Chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.

J Am Chem Soc. 2004 Dec 8; 126(48): 15658-9
Poelarends GJ, Serrano H, Johnson WH, Hoffman DW, Whitman CP

Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic beta-alpha-beta-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a kcat/Km value of 6.0 x 102 M-1 s-1. Pro-1 and Arg-75 are critical for both activities, and the pKa of Pro-1 was determined to be approximately 9.2 by a direct 15N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pKa value of Pro-1 are shared with trans-3-Chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water.

Bioorg Chem. 2004 Oct; 32(5): 376-92
Poelarends GJ, Whitman CP

The use of the soil fumigant Telone II, which contains a mixture of cis- and trans-1,3-dichloropropene, to control plant-parasitic nematodes is a common agricultural practice for maximizing yields of various crops. The effectiveness of Telone II is limited by the rapid turnover of the dichloropropenes in the soil due to the presence of bacterial catabolic pathways, which may be of recent origin. The characterization of three enzymes in these pathways, trans-3-Chloroacrylic acid dehalogenase (CaaD), cis-3-Chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD), has uncovered intriguing catalytic mechanisms as well as a fascinating evolutionary lineage for these proteins. Sequence comparisons and mutagenesis studies revealed that all three enzymes belong to the tautomerase superfamily. Tautomerase superfamily members with known structures are characterized by a beta-alpha-beta structural fold. Moreover, they have a conserved N-terminal proline, which plays an important catalytic role. Mechanistic, NMR, and pH rate studies of the two dehalogenases, coupled with a crystal structure of CaaD inactivated by 3-bromopropiolate, indicate that they use a general acid/base mechanism to catalyze the conversion of their respective isomer of 3-chloroacrylate to malonate semialdehyde. The reaction is initiated by the conjugate addition of water to the C-2, C-3 double bond and is followed by the loss of HCl. MSAD processes malonate semialdehyde to acetaldehyde, and is the first identified decarboxylase in the tautomerase superfamily. The catalytic mechanism is not well defined but the N-terminal proline plays a prominent role and may function as a general acid catalyst, similar to its role in CaaD and cis-CaaD. These are the first structural and mechanistic details for tautomerase superfamily members that catalyze either a hydration or a decarboxylation reaction, rather than a tautomerization reaction, in which Pro-1 serves as a general acid catalyst rather than as a general base catalyst. The available information on the 1,3-dichloropropene catabolic enzymes allows speculation on the possible evolutionary origins of their activities.

Biochemistry. 2004 Jun 8; 43(22): 7187-96
Poelarends GJ, Serrano H, Johnson WH, Whitman CP

The enzymes trans-3-Chloroacrylic acid dehalogenase (CaaD) and cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) represent the two major classes of bacterial, isomer-selective 3-Chloroacrylic acid dehalogenases. They catalyze the hydrolytic dehalogenation of either trans- or cis-3-haloacrylates to yield malonate semialdehyde, presumably through unstable halohydrin intermediates. In view of a proposed general acid/base mechanism for these enzymes, (R)- and (S)-oxirane-2-carboxylate were investigated as potential irreversible inhibitors. Only cis-CaaD is irreversibly inhibited in a time- and concentration-dependent manner and only by the (R)-enantiomer of oxirane-2-carboxylate. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These findings indicate that the inactivation process involves the initial formation of a reversibly bound enzyme-inhibitor complex at the active site followed by covalent modification. Mass spectral analysis of the inactivated cis-CaaD shows that Pro-1 is the site of modification. It has also been determined that Arg-70 and Arg-73 are required for covalent modification because incubation of either the R70A or R73A mutant with inhibitor does not result in enzyme alkylation. Studies of the pH dependence of the kinetic parameters of wild-type cis-CaaD reveal that a protonated group with a pK(a) of approximately 9.3 is essential for catalysis. The group is likely Pro-1, making it predominately a charged species under the conditions of the inactivation experiments. Two mechanisms could account for these observations. In one mechanism, the oxirane undergoes acid-catalyzed ring opening followed by alkylation of the conjugate base of Pro-1. Alternatively, the oxirane undergoes a nucleophilic substitution reaction where the conjugate base of Pro-1 functions as the nucleophile and an acid catalyst polarizes the carbon oxygen bond. The two arginine residues likely bind the carboxylate group and position the inhibitor in a favorable orientation for the alkylation reaction. These findings set the stage for a crystallographic analysis of the inactived enzyme to delineate further the roles of active site residues in both the inactivation process and the catalytic mechanism.

Biochemistry. 2004 Apr 13; 43(14): 4082-91
Azurmendi HF, Wang SC, Massiah MA, Poelarends GJ, Whitman CP, Mildvan AS

trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-Chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R(G)) disappeared in the spectrum of the inactive alphaR8A mutant (deltaH = 7.48 ppm, deltaN = 89.6 ppm), thereby assigning these resonances to alphaArg-11NepsilonH, and alphaArg-8NepsilonH, respectively. (1)H-(15)N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoic acid (3-CBA), a competitive inhibitor (K(I)(slope) = 0.35 +/- 0.06 mM), resulted in progressive downfield shifts of the alphaArg-8Nepsilon resonance yielding a K(D) = 0.77 +/- 0.44 mM, comparable to the (K(I)(slope), suggestive of active site binding. Increasing the pH of free CaaD to 8.9 at 5 degrees C resulted in the disappearance of all nine Arg-NepsilonH resonances due to base-catalyzed NepsilonH exchange. Saturating the enzyme with 3-CBA (16 mM) induced the reappearance of two NepsilonH signals, those of alphaArg-8 and alphaArg-11, indicating that the binding of the substrate analogue 3-CBA selectively slows the NepsilonH exchange rates of these two arginine residues. The kinetic and NMR data thus indicate that betaPro-1 is the acid catalyst, alphaGlu-52 is a reasonable candidate for the general base, and alphaArg-8 and alphaArg-11 participate in substrate binding and in stabilizing the aci-carboxylate intermediate in a Michael addition mechanism.

Biochemistry. 2004 Jan 27; 43(3): 759-72
Poelarends GJ, Serrano H, Person MD, Johnson WH, Murzin AG, Whitman CP

The gene encoding the cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization. Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of approximately 10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H NMR spectrum. The enzyme shares low but significant sequence similarity with the previously studied trans-3-Chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family. While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography. On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD. Subsequent site-directed mutagenesis experiments confirmed these predictions. The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD. Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1. The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism. The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence. The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.

Biochemistry. 2004 Jan 27; 43(3): 748-58
Wang SC, Johnson WH, Czerwinski RM, Whitman CP

4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate. The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction. These residues are also critical for the trans-3-Chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde. Recently, 3-bromo- and 3-chloropropiolate, the acetylene analogues of 3-haloacrylates, were characterized as potent irreversible inhibitors of CaaD due to the covalent modification of the catalytic proline. In view of these observations, an investigation of the behavior of 4-OT and YwhB with the 3-halopropiolates was undertaken. The results show that these compounds are potent irreversible inhibitors of 4-OT and YwhB with Pro-1 being the sole site of covalent modification by 3-bromopropiolate. The inactivation process could involve the enzyme-catalyzed addition of water to the 3-halopropiolate yielding an acyl halide, which would inactivate the enzyme or be initiated by the nucleophilic attack of Pro-1 at the C-3 position of the 3-halopropiolate in a Michael type reaction. The presence of the halogen along with Arg-11 could facilitate both reactions with the latter causing the polarization of the alpha,beta-unsaturated acids. The 3-halopropiolates are the first identified inhibitors of YwhB and confirm the importance of Pro-1 in its mechanism. In addition, the results set the stage for the use of these compounds as mechanistic probes of the primary as well as low level activities of 4-OT and YwhB.

J Biol Chem. 2004 Mar 19; 279(12): 11546-52
de Jong RM, Brugman W, Poelarends GJ, Whitman CP, Dijkstra BW

Isomer-specific 3-Chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-Chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2. Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.

Curr Opin Struct Biol. 2003 Dec; 13(6): 722-30
de Jong RM, Dijkstra BW

The dehalogenases make use of fundamentally different strategies to cleave carbon-halogen bonds. The structurally characterized haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoate-coenzyme A dehalogenases use substitution mechanisms that proceed via a covalent aspartyl intermediate. Recent X-ray crystallographic analysis of a haloalcohol dehalogenase and a trans-3-Chloroacrylic acid dehalogenase has provided detailed insight into a different intramolecular substitution mechanism and a hydratase-like mechanism, respectively. The available information on the various dehalogenases supports different views on the possible evolutionary origins of their activities.

J Am Chem Soc. 2003 Nov 26; 125(47): 14282-3
Wang SC, Johnson WH, Whitman CP

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-Chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates. The enzymes are highly selective for the (E)-isomer, and Pro-1 is critical for the activity while an arginine is likely required. Two mechanisms are proposed in which Pro-1 functions as a general base or a general acid catalyst and, along with the arginine, facilitates the Michael addition of water. Both mechanisms suggest an intriguing route for the evolution of the CaaD activity. One or more mutations could decrease the hydrophobic environment of the active site, which would make it more favorable for a hydrolytic reaction, thereby raising the pKa of Pro-1 and increasing the concentration of enzyme in the reactive form.

Biochemistry. 2003 Jul 29; 42(29): 8762-73
Wang SC, Person MD, Johnson WH, Whitman CP

Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-Chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.

Arch Biochem Biophys. 2002 Jun 1; 402(1): 1-13
Whitman CP

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-Chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.

Biodegradation. 2001; 12(1): 39-47
Ou LT, Thomas JE, Chung KY, Ogram AV

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)- 1,3-dichloropropene) was enriched from an enhanced soil. This mixed culture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presence of a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-Chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)- 1,3-D, respectively. (E)- 1,3-D was degraded faster than (Z)- 1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)- 1,3-D to (Z)- 1,3-D or the (Z) form to the (E) form did not occur.

J Bacteriol. 2001 Jul; 183(14): 4269-77
Poelarends GJ, Saunier R, Janssen DB

The genes (caaD1 and caaD2) encoding the trans-3-Chloroacrylic acid dehalogenase (CaaD) of the 1,3-dichloropropene-utilizing bacterium Pseudomonas pavonaceae 170 were cloned and heterologously expressed in Escherichia coli and Pseudomonas sp. strain GJ1. CaaD is a protein of 50 kDa that is composed of alpha-subunits of 75 amino acid residues and beta-subunits of 70 residues. It catalyzes the hydrolytic cleavage of the beta-vinylic carbon-chlorine bond in trans-3-Chloroacrylic acid with a turnover number of 6.4 s(-1). On the basis of sequence similarity, oligomeric structure, and subunit size, CaaD appears to be related to 4-oxalocrotonate tautomerase (4-OT). This tautomerase consists of six identical subunits of 62 amino acid residues and catalyzes the isomerization of 2-oxo-4-hexene-1,6-dioate, via hydroxymuconate, to yield 2-oxo-3-hexene-1,6-dioate. In view of the oligomeric architecture of 4-OT, a trimer of homodimers, CaaD is postulated to be a hexameric protein that functions as a trimer of alpha beta-dimers. The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the beta-subunit and Arg-11 of the alpha-subunit are active-site residues in CaaD. Pro-1 could act as the proton acceptor/donor, and Arg-11 is probably involved in carboxylate binding. Based on these findings, a novel dehalogenation mechanism is proposed for the CaaD-catalyzed reaction which does not involve the formation of a covalent enzyme-substrate intermediate.

Appl Microbiol Biotechnol. 1999 Nov; 52(6): 853-62
Katsivela E, Bonse D, Krüger A, Strömpl C, Livingston A, Wittich RM

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralize DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 uzilizes cis-3-chloroallylalcohol and cis-3-Chloroacrylic acid, whereas the metabolite trans-3-Chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l-1 led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD546 = 0.2). At the standard operating feed rate of 1.8 mmol h-1, the free biomass concentration was very low (OD546 = 0.04).