Characterization of 2-phenanthroate:CoA ligase from the sulfate-reducing, phenanthrene-degrading enrichment culture TRIP

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are chemically stable pollutants that are poorly degraded by microorganisms in anoxic sediments. The anaerobic degradation pathway of PAHs such as phenanthrene starts with a carboxylation reaction forming phenanthroic acid. In this study, we identified and characterized the next enzyme in the pathway, the 2-phenanthroate:CoA ligase involved in the ATP-dependent formation of 2-phenanthroyl-CoA from cell-free extracts of the sulfate-reducing enrichment culture TRIP grown anaerobically with phenanthrene. The identified gene sequence indicated that 2-phenanthroate:CoA ligase belongs to the phenylacetate:CoA ligase-like enzyme family. Based on the sequence, we predict a two-domain structure of the 2-phenanthroate:CoA ligase with a typical large N-terminal and a smaller C-terminal domain. Partial purification of 2-phenanthroate:CoA ligase allowed us to identify the coding gene in the genome. 2-Phenanthroate:CoA ligase gene was heterologously expressed in Escherichia coli. Characterization of the 2-phenanthroate:CoA ligase was performed using the partially purified enzyme from cell-free extract and the purified recombinant enzyme. Testing all possible phenanthroic acid isomers as substrate for the ligase reaction showed that 2-phenanthroic acid is the preferred substrate and only 3-phenanthroic acid can be utilized to a minor extent. This also suggests that the product of the prior carboxylase reaction is 2-phenanthroic acid. 2-Phenanthroate:CoA ligase has an optimal activity at pH 7.5 and is oxygen-insensitive, analogous to other aryl-CoA ligases. In contrast to aryl-Coenzyme A ligases reported in the literature, which need Mg2+ as cofactor, 2-phenanthroate:CoA ligase showed greatest activity with a combination of 5 mM MgCl2 and 5 mM KCl. Furthermore, a substrate inhibition was observed at ATP concentrations above 1 mM and the enzyme was also active with ADP.