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Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols.


ABSTRACT: Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.

SUBMITTER: Jouanneau Y 

PROVIDER: S-EPMC1489356 | BioStudies | 2006-01-01

REPOSITORIES: biostudies

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