Chi Li Yu, Ryan M. Summers, Yalan Li, Sujit Kumar Mohanty, Mani Subramanian, R. Marshall Pope

Understanding the genes and enzymes involved in caffeine metabolism can lead to applications such as production of methyl xanthines 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 (9.0g L-1) than the maximum tolerable levels of other reported bacteria. We took a novel approach towards 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 and 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.