Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls.
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 kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). 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 (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, 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 kcat 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 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.
Department of Biochemistry, Université Laval, Québec City, Québec G1K 7P4, Canada.
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D016956:Burkholderia cepacia / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005822:Genetic Vectors; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D011506:Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D005231:Fatty Acids, Unsaturated; D011506:Proteins; D011994:Recombinant Proteins; C085250:2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate; D011078:Polychlorinated Biphenyls; D006867:Hydrolases; C062907:2,6-dioxo-6-phenylhexa-3-enoate hydrolase; C092541:2-hydroxymuconate-semialdehyde hydrolase