Abstract
The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. EmM for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of α-helix (56%) with lesser amounts of random coil (21%), β-turn (13%) and β-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% α-helix, 33.7% random coil and 10.5% β-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60°C for 5 min; thereafter by cooling at 1°C min−1 to 45°C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60°C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330–350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.
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Notes
Erratum: The value reported for pI by Zhou et al. (2004) was incorrectly given as pH 6.42. The correct value is 5.70, compared with a predicted value of 5.54 given in UniProtKB/TrEMBL entry 085078 for the dehalogenase of Arthrobacter strain TM-1.
Abbreviations
- CD:
-
Circular dichroism
- 4-CB:
-
4-Chlorobenzoate
- 4-CBCoA:
-
4-Chloro-benzoyl CoA
- GdmCl:
-
Guanidinium chloride
- 4-HB:
-
4-Hydroxybenzoate
- 4-HBCoA:
-
4-Hydroxybenzoyl CoA
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Acknowledgements
L. Z. thanks the research community of the University of Greenwich for the award of a bursary. Particular thanks are due to Dr. Ronan O’Brien for his valuable assistance in securing CD spectra.
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Zhou, L., Poh, R.P.C., Marks, T.S. et al. Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1. Biodegradation 19, 65–75 (2008). https://doi.org/10.1007/s10532-007-9115-9
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DOI: https://doi.org/10.1007/s10532-007-9115-9