Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols

TL;DR

This study characterizes a broad-specificity dihydrodiol dehydrogenase from bacteria that metabolizes various PAH dihydrodiols, revealing its substrate range, kinetic properties, and potential regulatory mechanisms.

Contribution

The paper reports the cloning, purification, and detailed kinetic analysis of a novel PAH dihydrodiol dehydrogenase with broad substrate specificity and insights into its reaction mechanism.

Findings

Enzyme oxidizes dihydrodiols from multiple PAHs to catechols.

High catalytic efficiency with two- to four-ring dihydrodiols.

Reaction mechanism likely follows ordered Bi Bi model.

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]pyene, 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 $\mu$M, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 $\mu$M). At pH 7.0, the specificity constant ranged from (1.3 $\pm$ 0.1) x 106 M?1 s?1 with benz[a]anthracene 1,2-dihydrodiol to (20.0 $\pm$ 0.8) x 106 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.