부산대학교 도서관

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N -demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N -demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N 1 - and N 3 -specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N -demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N -demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications. • Crystal structures of bacterial N-demethylase A and B were determined in apo and substrate-bound states. • NdmAB forms a unique heterocomplex with 3:3 stoichiometry. • Structure of the binary complex between NdmA and a ferredoxin domain of NdmD was determined. • The structural and biochemical results provide a framework for engineering of caffeine degradation enzymes and thus increase the reaction products, methylxanthines, which are high-value biochemicals. Unlabelled Image [ABSTRACT FROM AUTHOR]