title: 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.
abstract: 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.
authors.affiliation: a Graduate Division of Nutritional and Environmental Sciences , University of Shizuoka , Shizuoka , Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Yamanashi
authors.forename: Seung-Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Hara
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Funa
mesh_terms: D000272:Adipates / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D007783:Lactones / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism*; D013011:Sorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014233:Tricarboxylic Acids / Q000378:metabolism
chemical_list: C558759:3-carboxymuconate; D000272:Adipates; D001426:Bacterial Proteins; D007783:Lactones; D011994:Recombinant Proteins; D014233:Tricarboxylic Acids; C005968:carboxymuconolactone; C027316:3-oxoadipic acid; C039879:muconolactone; D002265:Carboxylic Ester Hydrolases; C021927:3-oxoadipate enol-lactonase; D002262:Carboxy-Lyases; C019832:4-carboxymuconolactone decarboxylase; D013011:Sorbic Acid
keywords: Rhodococcus jostii RHA1; protocatechuate; β-ketoadipate pathway