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

Fingerprint

N Demethylating Oxidoreductases
Caffeine
Ferredoxins
Enzymes
Food and Beverages
Pseudomonas putida
Drug Industry
Enzyme Assays
Electron Transport
Biological Products
Cosmetics
Nitrogen
Soil
Carbon
X-Rays
Bacteria
Pharmaceutical Preparations

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. / Kim, Jun Hoe; Kim, Bong Heon; Brooks, Shelby; Kang, Seung Yeon; Summers, Ryan M.; Song, Hyun Kyu.

In: Journal of Molecular Biology, Vol. 431, No. 19, 06.09.2019, p. 3647-3661.

Research output: Contribution to journalArticle

Kim, Jun Hoe ; Kim, Bong Heon ; Brooks, Shelby ; Kang, Seung Yeon ; Summers, Ryan M. ; Song, Hyun Kyu. / Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. In: Journal of Molecular Biology. 2019 ; Vol. 431, No. 19. pp. 3647-3661.
@article{fadc596d2afe4ef88448f5879a988107,
title = "Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex",
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.",
keywords = "caffeine, N-demethylase, plant-type ferredoxin, rational protein engineering, reductase",
author = "Kim, {Jun Hoe} and Kim, {Bong Heon} and Shelby Brooks and Kang, {Seung Yeon} and Summers, {Ryan M.} and Song, {Hyun Kyu}",
year = "2019",
month = "9",
day = "6",
doi = "10.1016/j.jmb.2019.08.004",
language = "English",
volume = "431",
pages = "3647--3661",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "19",

}

TY - JOUR

T1 - Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

AU - Kim, Jun Hoe

AU - Kim, Bong Heon

AU - Brooks, Shelby

AU - Kang, Seung Yeon

AU - Summers, Ryan M.

AU - Song, Hyun Kyu

PY - 2019/9/6

Y1 - 2019/9/6

N2 - 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.

AB - 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.

KW - caffeine

KW - N-demethylase

KW - plant-type ferredoxin

KW - rational protein engineering

KW - reductase

UR - http://www.scopus.com/inward/record.url?scp=85071119666&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85071119666&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2019.08.004

DO - 10.1016/j.jmb.2019.08.004

M3 - Article

C2 - 31412262

AN - SCOPUS:85071119666

VL - 431

SP - 3647

EP - 3661

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 19

ER -