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J Biol Chem, Vol. 273, Issue 36, 22943-22949, September 4, 1998

Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls

Stephen Y. K. Seah, Giuseppe Terracina^§, Jeffrey T. Bolin^§, Peter Riebel, Victor Snieckus, and Lindsay D. Eltis

From the  Department of Biochemistry, Université Laval, Québec City, Québec G1K 7P4, Canada, the ^§ Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, and the  Guelph-Waterloo Center for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a k_cat of 5.0 (± 0.07) s¹ and a k_cat/K_m of 2.0 (± 0.08) × 10⁷ M¹ s¹ (100 mM phosphate, pH 7.5, 25 °C). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (k_cat/K_m of 2.0 (± 0.05) × 10⁶ M¹ s¹ and 9.0 (± 0.5) × 10⁶ M¹ s¹, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The k_cat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 Å, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.

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