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.
Chang
K H
KH
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Liang
P H
PH
Beck
W
W
Scholten
J D
JD
Dunaway-Mariano
D
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008670:Metals; D011994:Recombinant Proteins; D006867:Hydrolases; C042846:4-chlorobenzoate dehalogenase