Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PC Bs) by Burkholderia sp, strain LB400 (S. Y. K. Seah, G. Labbe, S. Nerdinger , M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701-1708, 2000). To determine whether this is also true in divergent biphenyl degrade rs, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hy perexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD(P6) hydrolyzed HOPDA with a k(cat)/K-m of 1.62 (+/- 0.03) x 10(7) M-1 s(-1) (100 mM phosphate [pH 7.5], 25 degreesC), which is within 70% of that of Bph(LB400). BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the p henyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety, However, the rhodococcal enzyme was si gnificantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydroly sis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, mo re specifically than HOPDA, Moreover, 4-Cl HOPDA competitively inhibited Bp hD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was o bserved in BphD(LB400). These results demonstrate that BphD is a key determ inant in the aerobic transformation of PCBs by divergent biphenyl degraders , but that there exists significant diversity in the specificity of these b iphenyl hydrolases.