Publications

• The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1.

  Zhou L., Marks T.S., Poh R.P., Smith R.J., Chowdhry B.Z., Smith A.R.

  4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit ... >> More

  4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity. << Less

  Biodegradation 15:97-109(2004) [PubMed] [EuropePMC]

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

  Chang K.H., Liang P.H., Beck W., Scholten J.D., Dunaway-Mariano D.

  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 follo ... >> More

  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. << Less

  Biochemistry 31:5605-5610(1992) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Crooks G.P., Copley S.D.

  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 chro ... >> More

  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. << Less

  Biochemistry 33:11645-11649(1994) [PubMed] [EuropePMC]

• Dehalogenation of 4-chlorobenzoate. Characterisation of 4-chlorobenzoyl-coenzyme A dehalogenase from Pseudomonas sp. CBS3.

  Loffler F., Lingens F., Muller R.

  Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobe ... >> More

  Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg2+ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 degrees C. The apparent Km value for 4-chlorobenzoyl-coenzyme A was 2.4-2.7 microM. Vmax was 1.1 x 10(-7) M sec-1 (2.2 mumol min-1 mg-1 of protein). The NH2-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase. << Less

  Biodegradation 6:203-212(1995) [PubMed] [EuropePMC]