A novel caffeine-degrading bacterium, Pseudomonas putida CBB5 was isolated from the soil by an enrichment procedure using caffeine as the sole source of carbon and nitrogen. CBB5 grew not only on caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources, but also on theophylline and 3-methylxanthine. Analyses of metabolites in spent media, resting cell suspensions, and crude cell extracts confirmed that CBB5 degraded caffeine via N-demethylation to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N-demethylated to xanthine via 7-methylxanthine. A previously unreported pathway for N-demethylation of theophylline to 1- and 3-methylxanthines, followed by further N-demethylation to xanthine, was also discovered in CBB5. A 240 kDa, Fe2+-dependent N-demethylase (Ndm) was purified from CBB5 by traditional chromatographic techniques. Ndm was composed of NdmA (40 kDa) and NdmB (35 kDa), which could not be resolved further. Ndm was active only in the presence of a partially purified protein which exhibited cytochrome c reductase activity (Ccr). Ccr transfered reducing equivalents from NAD(P)H to Ndm, which catalyzed an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Ndm displayed N-demethylation activity toward all substrates in the caffeine and theophylline metabolic pathways. Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-heme iron oxygenase base on its distinct absorption spectrum and significant identity of NdmA and NdmB sequences of other Rieske non-heme iron proteins. The ndmA- and ndmB- gene sequences were determined and cloned individually into the pET32a expression vector as C-terminal His-tagged proteins. Both NdmA-His and NdmB-His proteins were purified using a Ni-NTA column. NdmA-His, in conjunction with Ccr, was capable of N-demethylating caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively, suggesting that NdmA-His is a specific N-1-demethylase. Similarly, NdmB-His was determined to be a specific N-3-demethylase, as it was capable of N-demethylating caffeine, theophylline, theobromine, and 3-methylxanthine to paraxanthine, 1-methylxanthine, 7-methylxanthine, and xanthine, respectively. N-demethylation activity of 7-methylxanthine to xanthine (putative NdmC) co-eluted with the partially purified Ccr fraction. This is the first report of multiple, highly positional-specific, Rieske, non-heme iron N-demethylase enzymes for bacterial metabolism of purine alkaloids.