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- PDB-6icm: Pseudomonas putida CBB5 NdmA with ferredoxin domain of NdmD -

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Basic information

Entry
Database: PDB / ID: 6icm
TitlePseudomonas putida CBB5 NdmA with ferredoxin domain of NdmD
Components
  • Methylxanthine N1-demethylase NdmA
  • Oxidoreductase NdmD
KeywordsMETAL BINDING PROTEIN/OXIDOREDUCTASE / N-demethylase / Rieske oxygenase / reductase plant type ferredoxin / METAL BINDING PROTEIN-OXIDOREDUCTASE complex
Function / homology
Function and homology information


methylxanthine N1-demethylase / demethylase activity / alkaloid catabolic process / Oxidoreductases / 2 iron, 2 sulfur cluster binding / oxidoreductase activity / metal ion binding
Similarity search - Function
: / Vanillate O-demethylase oxygenase-like, C-terminal catalytic domain / Vanillate O-demethylase oxygenase C-terminal domain / : / 2Fe-2S ferredoxin, iron-sulphur binding site / 2Fe-2S ferredoxin-type iron-sulfur binding region signature. / Beta-grasp domain / 2Fe-2S iron-sulfur cluster binding domain / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulphur domain ...: / Vanillate O-demethylase oxygenase-like, C-terminal catalytic domain / Vanillate O-demethylase oxygenase C-terminal domain / : / 2Fe-2S ferredoxin, iron-sulphur binding site / 2Fe-2S ferredoxin-type iron-sulfur binding region signature. / Beta-grasp domain / 2Fe-2S iron-sulfur cluster binding domain / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily / Beta-grasp domain superfamily / 2Fe-2S ferredoxin-type iron-sulfur binding domain profile. / 2Fe-2S ferredoxin-type iron-sulfur binding domain / 2Fe-2S ferredoxin-like superfamily / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / Ubiquitin-like (UB roll) / Roll / Alpha Beta
Similarity search - Domain/homology
: / FE2/S2 (INORGANIC) CLUSTER / Methylxanthine N1-demethylase NdmA / Oxidoreductase NdmD
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 2.961 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 / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Methylxanthine N1-demethylase NdmA
B: Methylxanthine N1-demethylase NdmA
C: Methylxanthine N1-demethylase NdmA
D: Oxidoreductase NdmD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,86212
Polymers136,7974
Non-polymers1,0658
Water543
1
A: Methylxanthine N1-demethylase NdmA
B: Methylxanthine N1-demethylase NdmA
C: Methylxanthine N1-demethylase NdmA
D: Oxidoreductase NdmD
hetero molecules

A: Methylxanthine N1-demethylase NdmA
B: Methylxanthine N1-demethylase NdmA
C: Methylxanthine N1-demethylase NdmA
D: Oxidoreductase NdmD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)275,72524
Polymers273,5958
Non-polymers2,13016
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation12_565x,x-y+1,-z+1/61
Buried area22230 Å2
ΔGint-311 kcal/mol
Surface area90880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)118.265, 118.265, 455.692
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Methylxanthine N1-demethylase NdmA / 1-methylxanthine demethylase


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: Protein Oxidoreductase NdmD


Mass: 9659.104 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Gene: ndmD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H9N291

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Non-polymers , 4 types, 11 molecules

#3: Chemical
ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe2S2
#4: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe
#5: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / 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: 3.36 Å3/Da / Density % sol: 63.42 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1M bicine/tris pH 8.5, 0.02M monosaccharides, 10% w/v PEG 20000, 20% v/v PEG MME 500

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: BRUKER SMART 6500 / Detector: CCD / Date: Jan 30, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.95→50 Å / Num. obs: 40519 / % possible obs: 99.7 % / Redundancy: 17.1 % / Rrim(I) all: 0.122 / Net I/σ(I): 42.9
Reflection shellResolution: 2.961→3.067 Å

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementResolution: 2.961→42.46 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.98
RfactorNum. reflection% reflection
Rfree0.2612 1999 4.96 %
Rwork0.2036 --
obs0.2065 40319 99.62 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.961→42.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8737 0 13 3 8753
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0349031
X-RAY DIFFRACTIONf_angle_d1.13612227
X-RAY DIFFRACTIONf_dihedral_angle_d16.5275370
X-RAY DIFFRACTIONf_chiral_restr0.0581290
X-RAY DIFFRACTIONf_plane_restr0.0071597
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9615-3.03550.37411370.28232633X-RAY DIFFRACTION99
3.0355-3.11760.35241390.27252665X-RAY DIFFRACTION100
3.1176-3.20930.33291380.27312664X-RAY DIFFRACTION100
3.2093-3.31280.32421410.26162689X-RAY DIFFRACTION100
3.3128-3.43120.32711380.24992663X-RAY DIFFRACTION100
3.4312-3.56850.331420.2372712X-RAY DIFFRACTION100
3.5685-3.73080.32591400.22332698X-RAY DIFFRACTION100
3.7308-3.92740.23751430.21062724X-RAY DIFFRACTION100
3.9274-4.17320.22941410.17832715X-RAY DIFFRACTION100
4.1732-4.49510.23741430.16342742X-RAY DIFFRACTION100
4.4951-4.94690.19811440.16922766X-RAY DIFFRACTION100
4.9469-5.66130.22211460.17522789X-RAY DIFFRACTION100
5.6613-7.12710.26061480.20572836X-RAY DIFFRACTION100
7.1271-42.46420.2371590.19223024X-RAY DIFFRACTION99

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