Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Jun Hoe Kim, Bong Heon Kim, Shelby Brooks, Seung Yeon Kang, Ryan M. Summers, Hyun Kyu Song

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 N1- and N3-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.

Original languageEnglish
Pages (from-to)3647-3661
Number of pages15
JournalJournal of Molecular Biology
Volume431
Issue number19
DOIs
Publication statusPublished - 2019 Sep 6

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Keywords

  • caffeine
  • N-demethylase
  • plant-type ferredoxin
  • rational protein engineering
  • reductase

ASJC Scopus subject areas

  • Molecular Biology

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