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

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.

💡 Research Summary

The study focuses on the purification, kinetic characterization, and mechanistic analysis of a polycyclic aromatic hydrocarbon (PAH) dihydrodiol dehydrogenase (PDDH) from the PAH‑degrading bacterium Sphingomonas sp. strain CHY‑1. The authors cloned the bphB gene, which encodes the enzyme, into an expression vector that adds an N‑terminal hexahistidine tag. Recombinant protein was over‑produced in Escherichia coli BL21(DE3), purified by nickel affinity chromatography followed by size‑exclusion chromatography, and identified as a homotetramer with an apparent molecular mass of ~110 kDa.

Substrate specificity assays demonstrated that PDDH oxidizes cis‑1,2‑dihydrodiols derived from biphenyl and eight PAHs, including naphthalene, anthracene, phenanthrene, chrysene, benz