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Journal of Bacteriology, February 2003, p. 1253-1260, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1253-1260.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites

Frédéric H. Vaillancourt,1,2,{dagger} María-Amparo Haro,2,{ddagger} Nathalie M. Drouin,2,§ Zamil Karim,1 Halim Maaroufi,2 and Lindsay D. Eltis1,2*

Departments of Microbiology and Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3,1 Department of Biochemistry, Pavillon Marchand, Université Laval, Quebec City, Quebec G1K 7P4, Canada2

Received 3 October 2002/ Accepted 22 November 2002

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB ~ DHB ~ 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB ~ 6-Cl DHB ~ 3'-Cl DHB > DHB > 2'-Cl DHB ~ 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s-1, respectively). However, these isozymes had remarkably different apparent Km values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 µM for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.

* Corresponding author. Mailing address: Dept. of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., 300, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-0042. Fax: (604) 822-6041. E-mail: leltis{at}interchange.ubc.ca.

{dagger} Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

{ddagger} Present address: Biotools, B & M Labs, S.A., Valle de Tobalina, E-28021, Madrid, Spain.

§ Present address: Unité de Recherche en Vaccinologie, CHUQ, Pavillon CHUL, Quebec City, Quebec G1V 4G2, Canada.

Journal of Bacteriology, February 2003, p. 1253-1260, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1253-1260.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fortin, P. D., MacPherson, I., Neau, D. B., Bolin, J. T., Eltis, L. D. (2005). Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation. J. Biol. Chem. 280: 42307-42314 [Abstract] [Full Text]  
  • Fortin, P. D., Lo, A. T.-F., Haro, M.-A., Kaschabek, S. R., Reineke, W., Eltis, L. D. (2005). Evolutionarily Divergent Extradiol Dioxygenases Possess Higher Specificities for Polychlorinated Biphenyl Metabolites. J. Bacteriol. 187: 415-421 [Abstract] [Full Text]  
  • Ohnishi, K., Okuta, A., Ju, J., Hamada, T., Misono, H., Harayama, S. (2004). Molecular Breeding of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase for Enhanced Resistance to 3-Chlorocatechol. J Biochem 135: 305-317 [Abstract] [Full Text]  


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