+Open data
-Basic information
Entry | Database: PDB / ID: 6ick | ||||||
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Title | Pseudomonas putida CBB5 NdmA | ||||||
Components | Methylxanthine N1-demethylase NdmA | ||||||
Keywords | METAL BINDING PROTEIN / N-demethylase / Rieske oxygenase / non-heme iron center / caffeine degradation | ||||||
Function / homology | Function and homology information methylxanthine N1-demethylase / chlorophyllide a oxygenase activity / chlorophyll biosynthetic process / alkaloid catabolic process / demethylase activity / 2 iron, 2 sulfur cluster binding / metal ion binding Similarity search - Function | ||||||
Biological species | Pseudomonas putida (bacteria) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.952 Å | ||||||
Authors | Kim, J.H. / Kim, B.H. / Kang, S.Y. / Song, H.K. | ||||||
Funding support | Korea, Republic Of, 1items
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Citation | Journal: J Mol Biol / Year: 2019 Title: Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. Authors: Jun Hoe Kim / Bong Heon Kim / Shelby Brooks / Seung Yeon Kang / Ryan M Summers / Hyun Kyu Song / 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 ...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- and N-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. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 6ick.cif.gz | 235.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ick.ent.gz | 185.8 KB | Display | PDB format |
PDBx/mmJSON format | 6ick.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6ick_validation.pdf.gz | 470 KB | Display | wwPDB validaton report |
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Full document | 6ick_full_validation.pdf.gz | 481.9 KB | Display | |
Data in XML | 6ick_validation.xml.gz | 45.8 KB | Display | |
Data in CIF | 6ick_validation.cif.gz | 68.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ic/6ick ftp://data.pdbj.org/pub/pdb/validation_reports/ic/6ick | HTTPS FTP |
-Related structure data
Related structure data | 6iclC 6icmC 6icnC 6icoC 6icpC 6icqC C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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Unit cell |
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-Components
#1: Protein | Mass: 42379.398 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: ndmA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H9N289, methylxanthine N1-demethylase #2: Chemical | #3: Chemical | #4: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 3.24 Å3/Da / Density % sol: 62.02 % |
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Crystal grow | Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 0.2M ammonium citrate/citric acid pH 7.5, 15% w/v PEG 3350, 20% v/v 2-propanol |
-Data collection
Diffraction | Mean temperature: 100 K |
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Diffraction source | Source: SYNCHROTRON / Site: PAL/PLS / Beamline: 5C (4A) / Wavelength: 0.9795 Å |
Detector | Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 23, 2016 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 0.9795 Å / Relative weight: 1 |
Reflection | Resolution: 1.95→50 Å / Num. obs: 119695 / % possible obs: 99.9 % / Redundancy: 8.4 % / Rrim(I) all: 0.083 / Net I/σ(I): 26.2 |
Reflection shell | Resolution: 1.952→2.022 Å |
-Processing
Software |
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.952→38.802 Å / SU ML: 0.15 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 19.36
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 1.952→38.802 Å
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Refine LS restraints |
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LS refinement shell |
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