A tale of two enzymes : probing the aromatic hydrocarbon metabolism of Methylibium petroleiphilum, a degrader of petroleum groundwater pollutants, with the 4-oxalocrotonate tautomerase enzymes

Abstract

The cleanup of petroleum groundwater contamination is a major concern for the United States and as microbial bioremediation gains popularity the improvement of this method requires further research into how these microbes degrade water-soluble petroleum pollutants. Methylibium petroleiphilum (M. petroleiphilum) is one such bacterium of special interest, because it can metabolize a wide range of gasoline contaminants. The primary goal of this research is to investigate M. petroleiphilum's capacity for metabolizing different aromatic hydrocarbon compounds from petroleum pollution. The source of this unusual ability is hypothesized to stem from two seemingly redundant aromatic hydrocarbon-degrading operons, wherein the two operons would catabolize different petroleum contaminants and thus offer versatility to the bacteria. In this study, in vitro biochemical techniques probe two homologous proteins, specifically the 4-oxalocrotonate tautomerase (4OT) enzymes, within each operon. The 4OT family is well characterized and these proteins are especially good candidates for this research due to their ease of expression, purification, and robust catalytic properties. Furthermore, the 4OT family is known to be promiscuous with respect to substrate preference and catalysis. Also, the sequence identity between Tautomerase I and II is the lowest of all redundant aromatic hydrocarbon degrading enzymes, which suggests that these enzymes may show the most significant distinctions in substrate preference. As such, each enzyme has been recombinantly expressed and purified to enable detailed molecular-level characterization. The structure of each enzyme has been solved, using x-ray crystallography, and comparison of which provides insight into their potential functional differences. Kinetic analysis of two petroleum metabolites, specifically those of benzene and toluene, were measured for each enzyme. These results also suggest substrate preferences. The results of this research provide insight into the metabolism of petroleum groundwater pollutants, specifically the aromatic hydrocarbons, by Methylibium petroleiphilum.