title: Rapid identification and quantitative validation of a caffeine-degrading pathway in Pseudomonas sp. CES.
abstract: Understanding the genes and enzymes involved in caffeine metabolism can lead to applications such as production of methylxanthines and environmental waste remediation. Pseudomonas sp. CES may provide insights into these applications, since this bacterium degrades caffeine and thrives in concentrations of caffeine that are three times higher (9.0 g L(-1)) than the maximum tolerable levels of other reported bacteria. We took a novel approach toward identifying the enzymatic pathways in Pseudomonas sp. CES that metabolize caffeine, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities. Here we describe an optimized, rapid alternative strategy based on multiplexed LC-MS/MS assays and show its application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison of proteomes from bacteria grown in the absence and presence of caffeine, the latter condition of which was found to have a highly induced capacity for caffeine degradation. Comparisons were made using stable isotope dimethyl labeling, differences in the abundance of particular proteins were substantiated by reciprocal labeling experiments, and the role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Overall, multiple new components of a N-demethylase system were identified that resulted in rapid pathway validation and gene isolation using this new approach.
authors.affiliation: Proteomics Facility, University of Iowa , 355 EMRB, Iowa City, Iowa 52242, United States.
authors.forename: Chi Li
authors.initials: CL
authors.lastname: Yu
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Summers
authors.forename: Yalan
authors.initials: Y
authors.lastname: Li
authors.forename: Sujit Kumar
authors.initials: SK
authors.lastname: Mohanty
authors.forename: Mani
authors.initials: M
authors.lastname: Subramanian
authors.forename: R Marshall
authors.initials: RM
authors.lastname: Pope
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002110:Caffeine / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D002110:Caffeine; D010089:Oxidoreductases, N-Demethylating
keywords: LC-MS/MS; Pseudomonas sp. CES; ReDi; caffeine metabolism; protein identification; stable isotope dimethyl labeling