Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex
- Authors
- Kim, Jun Hoe; Kim, Bong Heon; Brooks, Shelby; Kang, Seung Yeon; Summers, Ryan M.; Song, Hyun Kyu
- Issue Date
- 6-9월-2019
- Publisher
- ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
- Keywords
- caffeine; N-demethylase; plant-type ferredoxin; rational protein engineering; reductase
- Citation
- JOURNAL OF MOLECULAR BIOLOGY, v.431, no.19, pp.3647 - 3661
- Indexed
- SCIE
SCOPUS
- Journal Title
- JOURNAL OF MOLECULAR BIOLOGY
- Volume
- 431
- Number
- 19
- Start Page
- 3647
- End Page
- 3661
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/62898
- DOI
- 10.1016/j.jmb.2019.08.004
- ISSN
- 0022-2836
- Abstract
- 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. (C) 2019 Elsevier Ltd. All rights reserved.
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