In vitro reconstitution of the catabolic reactions catalyzed by PcaHG, PcaB, and PcaL: the protocatechuate branch of the β-ketoadipate pathway in Rhodococcus jostii RHA1.

The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.