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- PDB-6icl: Pseudomonas putida CBB5 NdmB -

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Basic information

Entry
Database: PDB / ID: 6icl
TitlePseudomonas putida CBB5 NdmB
ComponentsMethylxanthine N3-demethylase NdmB
KeywordsMETAL BINDING PROTEIN / N-demethylase / Rieske oxygenase / non-heme iron center / caffeine degradation
Function / homology
Function and homology information


methylxanthine N3-demethylase / alkaloid catabolic process / demethylase activity / 2 iron, 2 sulfur cluster binding / oxidoreductase activity / metal ion binding / cytoplasm
Similarity search - Function
Vanillate O-demethylase oxygenase-like, C-terminal catalytic domain / Vanillate O-demethylase oxygenase C-terminal domain / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily
Similarity search - Domain/homology
: / FE2/S2 (INORGANIC) CLUSTER / Methylxanthine N3-demethylase NdmB
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 2.1 Å
AuthorsKim, J.H. / Kim, B.H. / Kang, S.Y. / Song, H.K.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (Korea)2016R1E1A1A01942623 Korea, Republic Of
CitationJournal: 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
DepositionSep 6, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Methylxanthine N3-demethylase NdmB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,3013
Polymers43,0701
Non-polymers2322
Water2,810156
1
A: Methylxanthine N3-demethylase NdmB
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)259,80818
Polymers258,4186
Non-polymers1,39012
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
crystal symmetry operation10_555-y,-x,-z+1/21
crystal symmetry operation11_555-x+y,y,-z+1/21
crystal symmetry operation12_555x,x-y,-z+1/21
Buried area18490 Å2
ΔGint-249 kcal/mol
Surface area82410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)98.553, 98.553, 174.441
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number182
Space group name H-MP6322

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Components

#1: Protein Methylxanthine N3-demethylase NdmB / 3-methylxanthine demethylase


Mass: 43069.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: ndmB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H9N290, methylxanthine N3-demethylase
#2: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster


Mass: 175.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe2S2
#3: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 156 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.84 Å3/Da / Density % sol: 56.68 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1M Tris-HCl pH 8.5, 0.5M NaCl, 5.36% w/v PEG 8000, 20.1% v/v MPD

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PAL/PLS / Beamline: 5C (4A) / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 17, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.1→50 Å / Num. obs: 30303 / % possible obs: 94.6 % / Redundancy: 12.1 % / Rrim(I) all: 0.062 / Net I/σ(I): 60
Reflection shellResolution: 2.1→2.168 Å

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementResolution: 2.1→41.452 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 22.15
RfactorNum. reflection% reflection
Rfree0.2217 1942 6.7 %
Rwork0.1955 --
obs0.1973 28968 95.57 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.1→41.452 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2740 0 5 156 2901
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0122825
X-RAY DIFFRACTIONf_angle_d1.1373831
X-RAY DIFFRACTIONf_dihedral_angle_d15.9511685
X-RAY DIFFRACTIONf_chiral_restr0.058410
X-RAY DIFFRACTIONf_plane_restr0.006496
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.0925-2.14490.28961270.24691722X-RAY DIFFRACTION87
2.1449-2.20280.31571280.22671773X-RAY DIFFRACTION91
2.2028-2.26770.24521360.22941812X-RAY DIFFRACTION92
2.2677-2.34090.26731330.21641847X-RAY DIFFRACTION93
2.3409-2.42450.24741310.21091860X-RAY DIFFRACTION95
2.4245-2.52160.27281350.21041937X-RAY DIFFRACTION96
2.5216-2.63630.24431390.21091902X-RAY DIFFRACTION96
2.6363-2.77530.21081380.20721950X-RAY DIFFRACTION98
2.7753-2.94910.20721400.21211966X-RAY DIFFRACTION98
2.9491-3.17670.26391430.19161994X-RAY DIFFRACTION98
3.1767-3.49630.22041410.19081988X-RAY DIFFRACTION99
3.4963-4.00180.1971480.17692042X-RAY DIFFRACTION100
4.0018-5.04050.19281480.16552079X-RAY DIFFRACTION100
5.0405-41.46040.211550.20852154X-RAY DIFFRACTION96

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