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- PDB-6icq: Pseudomonas putida CBB5 NdmA QL mutant with theobromine -

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Basic information

Entry
Database: PDB / ID: 6icq
TitlePseudomonas putida CBB5 NdmA QL mutant with theobromine
ComponentsMethylxanthine N1-demethylase NdmA
KeywordsMETAL BINDING PROTEIN / N-demethylase / Rieske oxygenase / non-heme iron center / caffeine degradation
Function / homology
Function and homology information


methylxanthine N1-demethylase / alkaloid catabolic process / demethylase activity / 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 / 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
THEOBROMINE / : / FE2/S2 (INORGANIC) CLUSTER / Methylxanthine N1-demethylase NdmA
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 1.9 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 ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)128,31412
Polymers127,0783
Non-polymers1,2359
Water14,970831
1
A: Methylxanthine N1-demethylase NdmA
B: Methylxanthine N1-demethylase NdmA
C: Methylxanthine N1-demethylase NdmA
hetero molecules

A: Methylxanthine N1-demethylase NdmA
B: Methylxanthine N1-demethylase NdmA
C: Methylxanthine N1-demethylase NdmA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,62724
Polymers254,1566
Non-polymers2,47118
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_556y,x,-z+11
Buried area20880 Å2
ΔGint-277 kcal/mol
Surface area83480 Å2
MethodPISA
Unit cell
Length a, b, c (Å)135.946, 135.946, 155.637
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

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


Mass: 42359.410 Da / Num. of mol.: 3 / Mutation: N282Q,F286L
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 ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster


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


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe
#4: Chemical ChemComp-37T / THEOBROMINE / 3,7-DIMETHYLXANTHINE / 3,7-DIMETHYLPURINE-2,6-DIONE / Theobromine


Mass: 180.164 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C7H8N4O2 / Comment: alkaloid*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 831 / 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: 3.27 Å3/Da / Density % sol: 62.35 %
Crystal growTemperature: 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

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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: Oct 13, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.9→50 Å / Num. obs: 132397 / % possible obs: 99 % / Redundancy: 7.2 % / Rrim(I) all: 0.101 / Net I/σ(I): 37.1
Reflection shellResolution: 1.9→1.96 Å

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementResolution: 1.9→35.041 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 19.64
RfactorNum. reflection% reflection
Rfree0.1963 2000 1.53 %
Rwork0.1697 --
obs0.1701 130758 98.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.9→35.041 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8166 0 0 831 8997
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0118424
X-RAY DIFFRACTIONf_angle_d1.03211448
X-RAY DIFFRACTIONf_dihedral_angle_d15.7554987
X-RAY DIFFRACTIONf_chiral_restr0.0631197
X-RAY DIFFRACTIONf_plane_restr0.0061494
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8924-1.93980.30211380.24448840X-RAY DIFFRACTION96
1.9398-1.99220.23751400.19629073X-RAY DIFFRACTION99
1.9922-2.05080.22291420.17579097X-RAY DIFFRACTION99
2.0508-2.1170.21931430.1759112X-RAY DIFFRACTION99
2.117-2.19270.18421420.15919163X-RAY DIFFRACTION99
2.1927-2.28040.19151390.17499090X-RAY DIFFRACTION99
2.2804-2.38420.19591420.16939160X-RAY DIFFRACTION99
2.3842-2.50990.22111470.17619179X-RAY DIFFRACTION99
2.5099-2.66710.24221450.18299218X-RAY DIFFRACTION99
2.6671-2.87290.21941390.18739256X-RAY DIFFRACTION99
2.8729-3.16190.21931450.18189258X-RAY DIFFRACTION100
3.1619-3.6190.19921460.17239331X-RAY DIFFRACTION100
3.619-4.5580.15871470.14749372X-RAY DIFFRACTION100
4.558-35.04660.17371450.16129609X-RAY DIFFRACTION99

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