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• W2034321085 abstract "dl-2-Haloacid dehalogenase fromPseudomonas sp. 113 (dl-DEX 113) catalyzes the hydrolytic dehalogenation of d- andl-2-haloalkanoic acids, producing the correspondingl- and d-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (l-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of dl-DEX 113. When a single turnover reaction of dl-DEX 113 was carried out with a large excess of the enzyme in H218O with a 10 times smaller amount of the substrate, either d- or l-2-chloropropionate, the major product was found to be18O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H218O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained 18O. These results indicate that the H218O of the solvent directly attacks the α-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate. dl-2-Haloacid dehalogenase fromPseudomonas sp. 113 (dl-DEX 113) catalyzes the hydrolytic dehalogenation of d- andl-2-haloalkanoic acids, producing the correspondingl- and d-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (l-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of dl-DEX 113. When a single turnover reaction of dl-DEX 113 was carried out with a large excess of the enzyme in H218O with a 10 times smaller amount of the substrate, either d- or l-2-chloropropionate, the major product was found to be18O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H218O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained 18O. These results indicate that the H218O of the solvent directly attacks the α-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate. Various enzymes catalyzing hydrolytic dehalogenation of organohalogen compounds have been isolated and characterized (1Fetzner S. Lingens F. Microbiol. Rev. 1994; 58: 641-685Crossref PubMed Google Scholar, 2Janssen D.B. Pries F. van der Ploeg J.R. Annu. Rev. Microbiol. 1994; 48: 163-191Crossref PubMed Scopus (147) Google Scholar, 3Slater J.H. Ratledge C. Biochemistry of Microbial Degradation. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 379-421Crossref Google Scholar). These enzymes include 2-haloacid dehalogenases (EC 3.8.1.2), haloacetate dehalogenases (EC 3.8.1.3), haloalkane dehalogenases (EC3.8.1.5), and 4-chlorobenzoyl-CoA dehalogenases (EC 3.8.1.6). 2-Haloacid dehalogenases are further classified into three groups based on their substrate specificities (4Soda K. Kurihara T. Liu J.-Q. Nardi-Dei V. Park C. Miyagi M. Tsunasawa S. Esaki N. Pure Appl. Chem. 1996; 68: 2097-2103Crossref Scopus (15) Google Scholar). l-2-Haloacid dehalogenase (l-DEX) 1The abbreviations used are: dl-DEX, dl-2-haloacid dehalogenase; l-DEX, l-2-haloacid dehalogenase; d-DEX, d-2-haloacid dehalogenase; dl-DEX 113, dl-DEX from Pseudomonas sp. 113; l-DEX YL, l-DEX from Pseudomonas sp. YL; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketonespecifically acts on l-2-haloalkanoic acids, and the corresponding d-2-hydroxyalkanoic acids are produced.d-2-Haloacid dehalogenase (d-DEX) catalyzes the conversion of d-2-hydroxyalkanoic acids intol-2-hydroxyalkanoic acids. dl-2-Haloacid dehalogenase (dl-DEX) dehalogenates both d- andl-2-haloalkanoic acids, and the correspondingl- and d-2-hydroxyalkanoic acids are produced.dl-DEX is similar to racemases and epimerases in that it acts indiscriminately on the chiral center of both d- andl-enantiomers. However, this enzyme is unique in that it catalyzes a chemical conversion on the chiral centers of both enantiomers. Thus far, the reaction mechanisms of l-DEX fromPseudomonas sp. YL (l-DEX YL) (5Liu J.-Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Liu J.-Q. Kurihara T. Miyagi M. Tsunasawa S. Nishihara M. Esaki N. Soda K. J. Biol. Chem. 1997; 272: 3363-3368Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7Li Y.-F. Hata Y. Fujii T. Hisano T. Nishihara M. Kurihara T. Esaki N. J. Biol. Chem. 1998; 273: 15035-15044Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (8Verschueren K.H.G. Seljee F. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Nature. 1993; 363: 693-698Crossref PubMed Scopus (422) Google Scholar, 9Pries F. Kingma J. Pentenga M. van Pouderoyen G. Jeronimus-Stratingh C.M. Bruins A.P. Janssen D.B. Biochemistry. 1994; 33: 1242-1247Crossref PubMed Scopus (98) Google Scholar, 10Pries F. Kingma J. Krooshof G. Jeronimus-Stratingh C. Bruins A. Janssen D. J. Biol. Chem. 1995; 270: 10405-10411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and 4-chlorobenzoyl-CoA dehalogenases from Pseudomonas sp. strain CBS3 (11Yang G. Liang P.-H. Dunaway-Mariano D. Biochemistry. 1994; 33: 8527-8531Crossref PubMed Scopus (51) Google Scholar, 12Benning M. Taylor K. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar) and Arthrobacter sp. 4-CB1 (13Crooks G.P. Xu L. Barkley R.M. Copley S.D. J. Am. Chem. Soc. 1995; 117: 10791-10798Crossref Scopus (30) Google Scholar) have been analyzed. Their reactions proceed as shown in Fig.1 A. Each of these dehalogenases has an acidic amino acid residue whose carboxylate group attacks the carbon atom of the substrate to which the halogen atom is bound. Asp10 of l-DEX YL, Asp124 of haloalkane dehalogenase from X. autotrophicus GJ10, and Asp145 of 4-chlorobenzoyl-CoA dehalogenase fromPseudomonas sp. strain CBS3 were identified to play this essential role for respective enzymes. The ester intermediates produced in the course of these reactions are subsequently hydrolyzed releasing the products and restoring the carboxylate groups of the enzymes. These were confirmed by chemical modification, site-directed mutagenesis, mass spectrometry, and x-ray crystallographical analysis (5Liu J.-Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Liu J.-Q. Kurihara T. Miyagi M. Tsunasawa S. Nishihara M. Esaki N. Soda K. J. Biol. Chem. 1997; 272: 3363-3368Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7Li Y.-F. Hata Y. Fujii T. Hisano T. Nishihara M. Kurihara T. Esaki N. J. Biol. Chem. 1998; 273: 15035-15044Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Verschueren K.H.G. Seljee F. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Nature. 1993; 363: 693-698Crossref PubMed Scopus (422) Google Scholar, 9Pries F. Kingma J. Pentenga M. van Pouderoyen G. Jeronimus-Stratingh C.M. Bruins A.P. Janssen D.B. Biochemistry. 1994; 33: 1242-1247Crossref PubMed Scopus (98) Google Scholar, 10Pries F. Kingma J. Krooshof G. Jeronimus-Stratingh C. Bruins A. Janssen D. J. Biol. Chem. 1995; 270: 10405-10411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Yang G. Liang P.-H. Dunaway-Mariano D. Biochemistry. 1994; 33: 8527-8531Crossref PubMed Scopus (51) Google Scholar, 12Benning M. Taylor K. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar, 13Crooks G.P. Xu L. Barkley R.M. Copley S.D. J. Am. Chem. Soc. 1995; 117: 10791-10798Crossref Scopus (30) Google Scholar). dl-DEXs have been purified from Pseudomonas sp. 113 (dl-DEX 113) (14Motosugi K. Esaki N. Soda K. J. Bacteriol. 1982; 150: 522-527Crossref PubMed Google Scholar), Pseudomonas putida PP3 (15Weightman A.J. Weightman A.L. Slater J.H. J. Gen. Microbiol. 1982; 128: 1755-1762PubMed Google Scholar), and Rhizobium sp. (16Leigh J.A. Skinner A.J. Cooper R.A. FEMS Microbiol. Lett. 1988; 49: 353-356Crossref Scopus (45) Google Scholar). However, none of the reaction mechanisms of these dl-DEXs have been studied, and it is unknown whether the reaction mechanism of dl-DEX is similar to that of other halidohydrolases (dehalogenases that catalyze the hydrolytic dehalogenation). We previously determined the primary structure of dl-DEX 113 (Fig.2), and found that it is similar to that of d-DEX from Pseudomonas putida AJ1 (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar). We also showed that dl-DEX 113 has a single and common catalytic site for both d- and l-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar). In the present study, we analyzed the reaction mechanism ofdl-DEX 113 by means of 18O incorporation experiments, and found that the reaction does not involve the formation of an enzyme-substrate ester intermediate. A water molecule is probably activated by a catalytic base of the enzyme, directly attacking the α-carbon of d- and l-2-haloalkanoic acids to displace the halogen atom (Fig. 1 B). This is the first example of an enzymatic dehalogenation that proceeds through the mechanism shown in Fig. 1 B. H218O (95–98%) was obtained from Cambridge Isotope Laboratories (Andover, MA) and Nippon Sanso (Tokyo, Japan). l- andd-2-chloropropionate were purchased from Sigma. Lysyl endopeptidase of Achromobacter lyticus M497–1 and trypsin (TPCK treated) were from Wako Industry Co., Ltd. (Osaka, Japan) and Sigma, respectively. All other chemicals were of analytical grade. RecombinantEscherichia coli JM109 cells harboring p4b (1Fetzner S. Lingens F. Microbiol. Rev. 1994; 58: 641-685Crossref PubMed Google Scholar) encodingdl-DEX 113 (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar) were cultivated at 37 °C for 14–18 h in a Luria-Bertani medium (1% polypeptone, 0.5% yeast extract, and 1% NaCl, pH 7.0) containing 150 μg/ml ampicillin and 0.2 mmisopropyl-1-thio-β-d-galactoside. The cells were collected by centrifugation, suspended in a 50 mm potassium phosphate buffer (pH 7.0), and disrupted by ultrasonic oscillation at 4 °C for 20 min with a Seiko Instruments ultrasonic disintegrator model 7500. The cell debris was removed by centrifugation. The supernatant solution was brought to 40% saturation with ammonium sulfate, and the precipitate was removed by centrifugation. The supernatant was applied to a Butyl Toyopearl 650M column, and elution was carried out with a linear gradient of 0–30% saturated ammonium sulfate in a 50 mm potassium phosphate buffer (pH 7.0). The active fractions were pooled and concentrated by ultrafiltration. For a single turnover experiment, 200 nmol of dl-DEX 113 in 50 μl of a 400 mmTris-H2SO4 buffer (pH 9.5) was lyophilized. The reaction was initiated by dissolving the dried enzyme in 50 μl of H218O containing 20 nmol of d- orl-2-chloropropionate (neutralized with NaOH), and the mixture was incubated at 30 °C for 24 h. The reaction mixtures were ultrafiltered, diluted 10-fold with 50% acetonitrile/H2O (1:1), and then introduced into the mass spectrometer using a Harvard Apparatus syringe infusion pump operating at 2 μl/min. The molecular mass of the produced lactate was measured with a PE-Sciex API III triple quadrupole mass spectrometer equipped with an ionspray ion source in the negative ion mode (Sciex, Thornhill, Ontario, Canada). Lyophilized 10 nmol of dl-DEX 113, 1.2 μmol of d- or l-2-chloropropionate (neutralized with NaOH), and 1.25 μmol of Tris-H2SO4 (pH 9.5) were mixed in 50 μl of H218O and incubated at 30 °C for 24 h. The enzyme was inactivated by incubating the reaction mixture for 10 min at 80 °C and then denatured by the addition of 100 μl of 5 m urea in 100 mmTris-H2SO4 (pH 7.5). Subsequently, the volume was adjusted to 500 μl with 120 mmTris-H2SO4 (pH 7.5) in order to reduce the urea concentration to 1 m and the pH to approximately 8.0.dl-DEX 113 in this solution was digested with 5 μg of TPCK-treated trypsin at 37 °C for 12 h. To digest dl-DEX 113 in H218O, the protein was denatured by the addition of 100 μl of 3 murea in 100 mm Tris-H2SO4 (pH 7.0) prepared with H218O. Thereafter, 150 μl of 100 mm Tris-H2SO4 (pH 7.5) containing 5 μg of trypsin prepared with H218O was added to this solution and incubated at 37 °C for 12 h. Lyophilized 10 nmol of dl-DEX 113, 1.2 μmol of d- or l-2-chloropropionate (neutralized with NaOH), and 2.5 μmol of Tris-H2SO4 (pH 9.5) were mixed in 50 μl of H218O and incubated at 30 °C for 24 h. The enzyme was inactivated by incubating the reaction mixture at 80 °C for 10 min, denatured with 8 m urea, and subsequently digested with 825 pmol of lysyl endopeptidase at 37 °C for 12 h. To digest the enzyme in H218O, 10 nmol ofdl-DEX 113, 1.0 μmol of d- orl-2-chloropropionate (neutralized with NaOH), and 20 μmol of Tris-H2SO4 (pH 9.5) were mixed in 50 μl of H218O and incubated at 37 °C for 24 h. A 20-μl aliquot was lyophilized and dissolved with 20 μl of H218O containing 8 m urea. After incubation at 37 °C for 1 h, 30 μl of 1 mTris-H2SO4 (pH 9.0) in H218O and 10 μl of 33 μm lysyl endopeptidase in H218O were added to this solution, and incubation was carried out at 37 °C for 12 h. The proteolytic digests of the enzyme were loaded onto a YMC-PackC4-AP column (100 × 1.0-mm inner diameter) (YMC Co., Kyoto, Japan) connected to the mass spectrometer and then eluted with a linear gradient of 0–80% acetonitrile in 0.05% trifluoroacetic acid over 80 min at a flow rate of 10 μl/min. A total ion current chromatogram was recorded in the single-quadrupole mode with a PE-Sciex API III mass spectrometer equipped with an ionspray ion source. The quadrupole was scanned from 300 to 2000 atomic mass units with a step size of 0.25 atomic mass units and a 0.5-ms dwell time per step. Ionspray voltage was set at 5 kV, and the orifice potential was 80 V. The molecular mass of each peptide was calculated with MacSpec software supplied by Sciex. Plasmid p4b (1Fetzner S. Lingens F. Microbiol. Rev. 1994; 58: 641-685Crossref PubMed Google Scholar) was mutagenized by the method of Kunkel et al. (18Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar). The mutant enzymes and synthetic mutagenic primers were as follows (the underlines indicate the mutagenized nucleotides): D181A, 5′-TCACGGATGGCCCTGAG-3′; D181R, 5′-TCACGGATGCGCCTGAG-3′; D181E, 5′-TCACGGATTTCCCTGAG-3′. Mutant enzymes were produced byE. coli BMH 71-18 mutS. dl-DEX 113, l-DEX YL (19Liu J.-Q. Kurihara T. Nardi-Dei V. Okamura T. Esaki N. Soda K. Biodegradation. 1995; 6: 223-227Crossref PubMed Scopus (10) Google Scholar), and l-DEX from Pseudomonas putida no. 109 (20Liu J.-Q. Kurihara T. Nardi-Dei V. Esaki N. Soda K. Bull. Inst. Chem. Res. Kyoto Univ. 1994; 72: 330-335Google Scholar) (final 0.45 mg/ml) were mixed with 1 m hydroxylamine in 1m Tris-H2SO4 (pH 9.0) in the presence or absence of 100 mml-2-chloropropionate and incubated at 30 °C for 60 min. After dialysis against a 50 mm potassium phosphate buffer (pH 7.5), the remaining activities of the enzyme were measured by the standard assay method as described below. dl-DEX 113 was assayed with 25 mmd- orl-2-chloropropionate as a substrate. The chloride ions released were measured spectrophotometrically (21Iwasaki I. Utsumi S. Hagino K. Ozawa T. Bull. Chem. Soc. Jpn. 1956; 29: 860-864Crossref Google Scholar). One unit of enzyme was defined as the amount of the enzyme that catalyzes the dehalogenation of 1 μmol of the substrate/min. The protein assay was done with a Bio-Rad protein assay kit. We conducted the single turnover reaction of dl-DEX 113 in H218O with d- or l-2-chloropropionate as a substrate, using an excess amount of the enzyme. We found that a majority of the lactate produced was labeled with 18O (Fig.3, A and B). This makes a clear contrast with our results on the l-DEX YL reaction, which proceeds through the mechanism involving an ester intermediate (Fig. 1 A) (5Liu J.-Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Only 10% of thed-lactate produced from l-2-chloropropionate byl-DEX YL was labeled with 18O (Fig.3 C). This suggests that in the dl-DEX 113 reaction an oxygen atom of the solvent water was directly incorporated into the product. While supporting the mechanism shown in Fig.1 B, this is not compatible with the mechanism in Fig.1 A, in which an oxygen atom of the solvent water is first incorporated into the enzyme. A multiple turnover reaction of dl-DEX 113 was carried out in H218O with d- orl-2-chloropropionate as a substrate. After completion of the reaction, the enzyme was digested with TPCK-treated trypsin, and the resulting peptide fragments were separated on a reversed phase column interfaced with an ionspray mass spectrometer as a detector. If the reaction proceeds through the mechanism in Fig. 1 B,18O should not be detected in the proteolytic fragments. The spectrometer was used in the single quadrupole mode, and the total ion current chromatogram was obtained as shown in Fig.4. Peaks 1–21 were assigned to peptides 144–151, 143–151, 67–76, 2–8, 286–298, 301–307, 9–21, 184–196, 108–119, 239–249, 152–162, 22–34, 211–228, 77–105, 273–283, 44–66, 163–180 (dimer), 197–210, 120–134, 255–272, and 35–43, respectively (Table I, Fig. 2). The molecular masses of all peptides were virtually indistinguishable from the predicted ones whether the reaction was conducted withd- or l-2-chloropropionate.Table IMolecular masses of proteolytic fragments of dl-DEX 113 incubated with substrates in H218OPeakFragment[M + H]+Predicted (average mass)Measuredl-CPA1-aDL-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin.d-CPA1-aDL-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin.1144–151933.99933.5933.82143–1511090.181089.51089.5367–761138.221137.51137.542–8851.04850.5850.85286–2981368.401367.81367.86301–307816.93816.5816.579–211465.571465.01465.08184–1961486.591486.01486.09108–1191263.391262.81262.810239–2491093.191092.81092.811152–1621201.411200.81200.81222–341545.691545.61545.913211–2282023.292022.72022.71477–1052919.262919.42919.115273–2831232.511231.81231.81644–662674.102674.42674.217163–180 (dimer)4165.724165.94166.218197–2101664.951665.51665.519120–1341830.101830.51830.520255–2722043.422043.22043.22135–431045.281046.01046.21-a DL-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin. Open table in a new tab Since peptides containing amino acid residues 1, 106–107, 135–142, 181–183, 229–238, 250–254, 284–285, and 299–300 were not found in the trypsin-digested sample, we also analyzed lysyl endopeptidase-digested enzyme by the same method. Peptides 120–142, 232–285, and 299–306 were identified, and their molecular masses were virtually indistinguishable from the predicted ones (TableII, Fig. 2). The molecular masses of the peptides containing amino acid residues 1, 106–107, 181–183, and 229–231 could not be measured.Table IIMolecular masses of proteolytic fragments of dl-DEX 113 incubated with substrates in H218OFragment[M + H]+Predicted (average mass)Measuredl-CPA2-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase.d-CPA2-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase.120–1422443.732443.72443.7232–2856023.056023.56023.3299–3061063.251062.81062.82-a dl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase. Open table in a new tab An oxygen atom of the catalytic carboxylate group of haloalkane dehalogenase fromX. autotrophicus GJ10 is rapidly replaced by an oxygen atom of the solvent water even in the absence of the substrate (9Pries F. Kingma J. Pentenga M. van Pouderoyen G. Jeronimus-Stratingh C.M. Bruins A.P. Janssen D.B. Biochemistry. 1994; 33: 1242-1247Crossref PubMed Scopus (98) Google Scholar). If this is the case for dl-DEX 113, 18O once incorporated into the acidic amino acid residue during the dehalogenation should have been replaced by the 16O of the solvent water during the treatment with trypsin or lysyl endopeptidase, which raised the possibility that the increase in the molecular mass of the peptides might not be detectable in the above experiments. To examine this possibility, we carried out the denaturation and trypsin digestion of the enzyme in H218O. If there is an oxygen atom (16O) in the enzyme which is readily exchangeable for an oxygen (18O) of the solvent water, an increase in the molecular mass of the proteolytic peptides is expected. The molecular masses of the proteolytic peptides, 2–8, 9–21, 22–34, 35–43, 44–66, 67–76, 77–105, 108–119, 120–134, 144–151, 152–162, 184–196, 197–210, 211–228, 239–249, 255–272, and 273–283 were approximately 4 Da higher than the predicted molecular masses in the system with either d- orl-2-chloropropionate as a substrate (TableIII, Fig. 2). A peptide containing amino acid residues 163–180 appeared as a dimeric form, and its molecular mass was approximately 8 Da higher than the predicted one. These increases are attributed to the incorporation of two 18O atoms into the α-carboxylate group of the C-terminal amino acid residue of each tryptic fragment. The molecular mass of peptide 301–307 derived from the C-terminal region of the enzyme was almost identical to the predicted value. Peptide fragments prepared with lysyl endopeptidase were also analyzed, and the molecular masses of peptides 120–142, 232–285, 286–298, and 299–306 were about 4 Da higher than the predicted ones (Table IV, Fig. 2). These show that the 18O of the solvent water was incorporated only into the α-carboxylate group of each peptide but not into the side chain carboxylate group.Table IIIMolecular masses of proteolytic fragments of dl-DEX 113 incubated with substrates in H218OPeakFragment[M + H]+Predicted (average mass)Measuredl-CPA3-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin in H218O.d-CPA3-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin in H218O.1144–151933.99937.8937.8267–761138.221142.01141.832–8851.04855.0855.049–211465.571469.21470.05301–307816.93816.8816.86184–1961486.591490.21490.67108–1191263.391267.01267.28239–2491093.191097.01097.09152–1621201.411205.21205.01022–341545.691549.21549.211211–2282023.292027.32026.91277–1052919.262923.52923.21335–431045.281049.21048.814273–2831232.511236.01236.21544–662674.102678.42677.816163–180 (dimer)4165.724173.64173.617197–2101664.951669.01669.818120–1341830.101833.91834.419255–2722043.422047.32047.33-a dl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with trypsin in H218O. Open table in a new tab Table IVMolecular masses of proteolytic fragments of dl-DEX 113 incubated with substrates in H218OFragment[M + H]+Predicted (average mass)Measuredl-CPA4-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase in H218O.d-CPA4-adl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase in H218O.120–1422443.732446.92447.2232–2856023.056027.76027.0286–2981368.401371.81371.9299–3061063.251066.71066.74-a dl-DEX 113 was incubated withl-2-chloropropionate (l-CPA) ord-2-chloropropionate (d-CPA) in H218O, and digested with lysyl endopeptidase in H218O. Open table in a new tab The molecular masses of peptides 1 (M), 106–107 (LK), 143 (R), 181–183 (DIR), and 229–231 (IRK) could not be determined in the above experiments. Therefore, we could not exclude the possibility that 18O was incorporated into Asp or Glu in these peptides. However, among these peptides, only peptide 181–183 contains an acidic residue, Asp181. We replaced Asp181with Ala, Arg, and Glu by site-directed mutagenesis to clarify whether Asp181 is involved in the catalytic reaction shown in Fig.1 A as Asp10 of l-DEX YL is. The activities of these mutant enzymes were similar to that of the wild-type enzyme (Table V), indicating that Asp181 is not essential for the catalysis.Table VActivities of wild-type and mutant dl-DEX 113EnzymesSpecific activity (units/mg)l-2-Chloropropionated-2-ChloropropionateWild-type1.071.01D181A1.241.26D181R1.100.98D181E1.061.00Specific activities of the crude extracts of the recombinant E. coli cells were measured with 25 mM l- ord-2-chloropropionate. Open table in a new tab Specific activities of the crude extracts of the recombinant E. coli cells were measured with 25 mM l- ord-2-chloropropionate. We previously found that hydroxylamine performs a nucleophilic attack on the active site aspartate residue (Asp10) of l-DEX YL (6Liu J.-Q. Kurihara T. Miyagi M. Tsunasawa S. Nishihara M. Esaki N. Soda K. J. Biol. Chem. 1997; 272: 3363-3368Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This inactivation was observed only in the presence of the substrate, and an ester intermediate formed from Asp10 and the substrate was thought to be a target of hydroxylamine. This was confirmed by mass spectrometric analysis of the inactivated enzyme, which showed that the modified Asp10 residue contained both hydroxylamine- and substrate-derived moieties. The inactivation of 4-chlorobenzoyl-CoA dehalogenase by hydroxylamine was also reported (13Crooks G.P. Xu L. Barkley R.M. Copley S.D. J. Am. Chem. Soc. 1995; 117: 10791-10798Crossref Scopus (30) Google Scholar). In contrast, no inactivation of dl-DEX 113 was observed (Table VI).Table VIEffect of hydroxylamine on dehalogenase activityActivityAddition ofHydroxylamine−++Substrate−−+Remaining activity (%)l-DEX YL1001020.06l-DEX from Pseudomonas sp. 109100970.20dl-DEX 113100100100 Open table in a new tab The reaction mechanism of dl-DEX 113 was studied by18O incorporation experiments and site-directed mutagenesis. Single turnover reactions carried out in H218O indicated that an oxygen atom of the solvent water is directly incorporated into the product (Fig. 3,A and B). We also found that an oxygen atom of the solvent water is not incorporated into the side chain carboxylate groups of the acidic amino acid residues of the enzyme in the dehalogenation reaction (except for Asp181, whose molecular mass could not be measured) (Tables Table I, Table II, Table III, Table IV). A site-directed mutagenesis experiment showed that Asp181 is not essential in the catalysis (Table V). These results are consistent with the general base mechanism shown in Fig. 1 B, but not with the mechanism shown in Fig. 1 A. This applies to the dehalogenations of both enantiomers of 2-haloalkanoic acids because the results obtained for both enantiomers were virtually the same. We previously reported thatdl-DEX 113 has a single and common catalytic site for bothl- and d-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar). This conclusion is supported by our present data showing that the enzymatic dehalogenations of both enantiomers proceed through the same mechanism as shown in Fig. 1 B. The reaction mechanisms of three kinds of halidohydrolases have been analyzed: haloalkane dehalogenase from X. autotrophicus GJ10 (8Verschueren K.H.G. Seljee F. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Nature. 1993; 363: 693-698Crossref PubMed Scopus (422) Google Scholar, 9Pries F. Kingma J. Pentenga M. van Pouderoyen G. Jeronimus-Stratingh C.M. Bruins A.P. Janssen D.B. Biochemistry. 1994; 33: 1242-1247Crossref PubMed Scopus (98) Google Scholar, 10Pries F. Kingma J. Krooshof G. Jeronimus-Stratingh C. Bruins A. Janssen D. J. Biol. Chem. 1995; 270: 10405-10411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), 4-chlorobenzoyl-CoA dehalogenases from Pseudomonassp. strain CBS3 (11Yang G. Liang P.-H. Dunaway-Mariano D. Biochemistry. 1994; 33: 8527-8531Crossref PubMed Scopus (51) Google Scholar, 12Benning M. Taylor K. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar), and Arthrobacter sp. 4-CB1 (13Crooks G.P. Xu L. Barkley R.M. Copley S.D. J. Am. Chem. Soc. 1995; 117: 10791-10798Crossref Scopus (30) Google Scholar) and l-DEX YL (5Liu J.-Q. Kurihara T. Miyagi M. Esaki N. Soda K. J. Biol. Chem. 1995; 270: 18309-18312Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Li Y.-F. Hata Y. Fujii T. Hisano T. Nishihara M. Kurihara T. Esaki N. J. Biol. Chem. 1998; 273: 15035-15044Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The reactions of these dehalogenases proceed as shown in Fig. 1 A. Although there are no similarities in their primary and tertiary structures (12Benning M. Taylor K. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar, 22Janssen D.B. Pries F. van der Ploeg J. Kazemier B. Terpstra P. Witholt B. J. Bacteriol. 1989; 171: 6791-6799Crossref PubMed Google Scholar, 23Babbitt P.C. Kenyon G.L. Martin B.M. Charest H. Slyvestre M. Scholten J.D. Chang K.H. Liang P.H. Dunaway-Mariano D. Biochemistry. 1992; 31: 5594-5604Crossref PubMed Scopus (189) Google Scholar, 24Nardi-Dei V. Kurihara T. Okamura T. Liu J.-Q. Koshikawa H. Ozaki H. Terashima Y. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 3375-3380Crossref PubMed Google Scholar, 25Verschueren K. Franken S. Rozeboom H. Kalk K. Dijkstra B. J. Mol. Biol. 1993; 232: 856-872Crossref PubMed Scopus (128) Google Scholar, 26Hisano T. Hata Y. Fujii T. Liu J.-Q. Kurihara T. Esaki N. Soda K. J. Biol. Chem. 1996; 271: 20322-20330Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), they resemble one another in their catalytic reactions, in which an essential ester intermediate is produced from the active site nucleophilic carboxylate and the substrate molecule. So far, no halidohydrolases have been shown to catalyze the reaction without the formation of the ester intermediate. Thus, dl-DEX 113 is unique in that its reaction does not involve the formation of an ester intermediate. Since d-DEX from P. putida AJ1 (27Barth P.T. Bolton L. Thomson J.C. J. Bacteriol. 1992; 174: 2612-2619Crossref PubMed Google Scholar) shows sequence similarity with dl-DEX 113 (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar), the reaction of d-DEX probably proceeds through the mechanism shown in Fig. 1 B. Although dl-DEX andl-DEX can catalyze the same reaction (hydrolysis ofl-2-haloalkanoic acids), our present data clearly show that the reaction mechanisms of dl-DEX and l-DEX are completely different from each other. Recently, we found that Glu69 and Asp194 are essential for the catalysis of dl-DEX 113 by site-directed mutagenesis (17Nardi-Dei V. Kurihara T. Park C. Esaki N. Soda K. J. Bacteriol. 1997; 179: 4232-4238Crossref PubMed Google Scholar). Several hydrolases such as aspartic proteases (28Hyland L.J. Tomaszek Jr., T.A. Meek T.D. Biochemistry. 1991; 30: 8454-8463Crossref PubMed Scopus (251) Google Scholar) and glycosyl hydrolases (29Marcotte E. Monzingo A. Ernst S. Brzezinski R. Robertus J. Nat. Struct. Biol. 1996; 3: 155-162Crossref PubMed Scopus (120) Google Scholar) have been shown to possess Glu or Asp as a catalytic base that activates a water molecule to attack the substrate molecule. In these enzymatic hydrolyses, ester intermediates are not produced. In this respect, dl-DEX 113 resembles these hydrolases, and Glu69 and/or Asp194 may be involved in the activation of a water molecule that attacks the α-carbon atom of the substrate. Further studies including crystallographic analysis of the enzyme are now being carried out to identify the active site residues and to clarify their roles in the catalysis." @default.
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• W2034321085 title "dl-2-Haloacid Dehalogenase fromPseudomonas sp. 113 Is a New Class of Dehalogenase Catalyzing Hydrolytic Dehalogenation Not Involving Enzyme-Substrate Ester Intermediate" @default.
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