Q-181. Sequence and Protein Expression of the Polyaromatic Hydrocarbon Degrading Enzyme 2-Hydroxy-2H-chromene-2-carboxylate Isomerase from Sphingomonas paucimobilis EPA505

A. K. Brown, T. A. Hughes;
Clemson Univ., Clemson, SC.

Polyaromatic hydrocarbons (PAHs) are one of the major environmental hazards found at EPA Superfund sites around the nation. PAHs are also listed on the EPA list of thirty-one priority chemicals, but unfortunately this group of chemicals is very stable and slow to break down in the environment. However, there are many microorganisms capable of degrading such compounds_one of which is Sphingomonas paucimobilis EPA505. This species is able to metabolize a vast array of PAHs, but its mechanisms are only poorly understood. In this study, both genomic and proteomic approaches have revealed the nature of the 2-hydroxy-2H-chromene-2-carboxylate isomerase enzyme. This enzyme is used in the upper catabolic pathway of PAH degradation for the conversion of 2-hydroxy-2H-chromene-2-carboxylic acid to trans-o-hydroxybenzylidene-pyruvic acid. The gene encoding the enzyme (nahD) was sequenced from a BAC library via subcloning and high-throughput shotgun techniques. Protein expression was then performed using pUR vector system and analyzing β-galactosidase activity. This research as well as future studies should lead to a successful bioremediation strategy for PAHs with S. paucimobilis in the environment.