Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26.
The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
Nagata
Y
Y
Department of Biotechnology, University of Tokyo, Japan. aynaga@hongo.ecc.u-tokyo.ac.jp
Miyauchi
K
K
Damborsky
J
J
Manova
K
K
Ansorgova
A
A
Takagi
M
M
D001673:Biodegradation, Environmental; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001556:Hexachlorocyclohexane / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D001556:Hexachlorocyclohexane; D006867:Hydrolases; C046137:haloalkane dehalogenase