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Journal of Bacteriology, March 2001, p. 1511-1516, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1511-1516.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Comparative Specificities of Two Evolutionarily Divergent Hydrolases Involved in Microbial Degradation of Polychlorinated Biphenyls

Stephen Y. K. Seah,1,2,dagger Geneviève Labbé,1,2 Stefan R. Kaschabek,3 Frank Reifenrath,3 Walter Reineke,3 and Lindsay D. Eltis1,2,*

Department of Biochemistry, Université Laval, Québec, Québec,1 and Department of Microbiology and Immunology, University of British Columbia, Vancouver,2 Canada, and Chemische Mikrobiologie, Bergische Universität---GH Wuppertal, Wuppertal, Germany3

Received 12 July 2000/Accepted 28 November 2000

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701-15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphDP6, was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphDP6 hydrolyzed HOPDA with a kcat/Km of 1.62 (± 0.03) × 107 M-1 s-1 (100 mM phosphate [pH 7.5], 25°C), which is within 70% of that of BphDLB400. BphDP6 was also similar to BphDLB400 in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphDP6 more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphDLB400. These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, B.C., Canada V6T 1Z3. Phone: (604) 822-0042. Fax: (604) 822-0010. E-mail: leltis{at}interchange.ubc.ca.

dagger Present address: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada.

Journal of Bacteriology, March 2001, p. 1511-1516, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1511-1516.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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