The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1.
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.
Zhou
Lihong
L
Medway School of Science, University of Greenwich at Medway, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK.
Marks
Trevor S
TS
Poh
Roy P C
RP
Smith
Rodney J
RJ
Chowdhry
Babur Z
BZ
Smith
Anthony R W
AR
D001173:Arthrobacter / Q000737:chemistry*; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D011549:Pseudomonas / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D006867:Hydrolases; C084333:4-chlorobenzoyl coenzyme A dehalogenase; C077846:4-halobenzoate-CoA ligase; D003066:Coenzyme A Ligases