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- PDB-9azo: Crystal structure of CHMS Dehydrogenase PmdC from Comamonas testo... -

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Basic information

Entry
Database: PDB / ID: 9azo
TitleCrystal structure of CHMS Dehydrogenase PmdC from Comamonas testosteroni bound to cofactor NADP
ComponentsPmdC
KeywordsOXIDOREDUCTASE / NADP cofactor / Pyrone dicarboxylic acid / lignin degradation
Function / homology4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase-like, C-terminal / Bacterial oxidoreductases, C-terminal / : / Gfo/Idh/MocA-like oxidoreductase, N-terminal / Oxidoreductase family, NAD-binding Rossmann fold / NAD(P)-binding domain superfamily / nucleotide binding / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / PmdC
Function and homology information
Biological speciesComamonas testosteroni ATCC 11996 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.34 Å
AuthorsRodrigues, A.V. / Moriarty, N.W. / Pereira, J.H. / Adams, P.D.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-AC0205CH11231 United States
Citation
Journal: J.Biol.Chem. / Year: 2024
Title: Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.
Authors: Rodrigues, A.V. / Moriarty, N.W. / Kakumanu, R. / DeGiovanni, A. / Pereira, J.H. / Gin, J.W. / Chen, Y. / Baidoo, E.E.K. / Petzold, C.J. / Adams, P.D.
#1: Journal: ACS Nano / Year: 2024
Title: Pore Engineering as a General Strategy to Improve Protein-Based Enzyme Nanoreactor Performance.
Authors: Seokmu Kwon / Michael P Andreas / Tobias W Giessen /
Abstract: Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, ...Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.
History
DepositionMar 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Database references / Structure summary / Category: citation / pdbx_entry_details
Item: _citation.journal_volume / _citation.title / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PmdC
B: PmdC
C: PmdC
D: PmdC
E: PmdC
F: PmdC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)216,63919
Polymers211,5066
Non-polymers5,13313
Water6,485360
1
A: PmdC
B: PmdC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,1816
Polymers70,5022
Non-polymers1,6794
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: PmdC
D: PmdC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,1816
Polymers70,5022
Non-polymers1,6794
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
E: PmdC
F: PmdC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,2777
Polymers70,5022
Non-polymers1,7755
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)79.151, 157.864, 95.011
Angle α, β, γ (deg.)90.000, 114.449, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11LYSLYSASNASNAA3 - 3173 - 317
d_12SO4SO4SO4SO4AG401
d_13NAPNAPNAPNAPAH402
d_21LYSLYSASNASNBB3 - 3173 - 317
d_22SO4SO4SO4SO4BI401
d_23NAPNAPNAPNAPBJ402
d_31LYSLYSASNASNCC3 - 3173 - 317
d_32SO4SO4SO4SO4CK401
d_33NAPNAPNAPNAPCL402
d_41LYSLYSASNASNDD3 - 3173 - 317
d_42SO4SO4SO4SO4DM401
d_43NAPNAPNAPNAPDN402
d_51LYSLYSASNASNEE3 - 3173 - 317
d_52SO4SO4SO4SO4EO401
d_53NAPNAPNAPNAPEP402
d_61LYSLYSASNASNFF3 - 3173 - 317
d_62SO4SO4SO4SO4FQ401
d_63NAPNAPNAPNAPFR402

NCS oper:
IDCodeMatrixVector
1given(0.999954733875, -0.00657423909277, -0.0068781961253), (-0.00655468329472, -0.999974422649, 0.00286184796006), (-0.006896834672, -0.00281663401805, -0.999972249737)-0.292217332369, -64.2966578939, -24.4522184951
2given(-0.640672361017, 0.404447898237, -0.652656742431), (0.421541277196, -0.525177671863, -0.739250542507), (-0.641749076703, -0.748739147226, 0.165975335404)17.6033208793, -67.527705303, -77.984020182
3given(-0.617205856422, -0.404019106796, 0.675148496363), (0.442511434539, 0.53126777839, 0.722452889777), (-0.650569412976, 0.744663084212, -0.149118509622)8.29319237118, -15.6513386323, -33.8410309189
4given(-0.617517120841, -0.403121243069, 0.675400524767), (-0.4607847052, -0.510500969305, -0.725993261533), (0.637455928612, -0.759527500309, 0.129490985604)-31.2435884927, -48.4121033342, -77.6728341416
5given(-0.629062577797, 0.39874482302, -0.667295166572), (-0.442489651012, 0.522099114706, 0.72911962199), (0.639126890346, 0.753933074309, -0.151992557381)-22.2072437335, 2.82064193546, -32.4385997112

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Components

#1: Protein
PmdC


Mass: 35250.941 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Comamonas testosteroni ATCC 11996 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q93PS4, Oxidoreductases
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: SO4
#3: Chemical
ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 360 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.58 Å3/Da / Density % sol: 52.41 %
Crystal growTemperature: 294.2 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 0.2 M ammonium sulfate, 0.1 M Bis-Tris, pH 5.5, 25% PEG3350
PH range: 5.0-6.0 / Temp details: ambient temperature

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Data collection

Diffraction
IDMean temperature (K)Crystal-IDSerial crystal experiment
1801N
2801N
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONNSLS-II 17-ID-211
SYNCHROTRONALS 8.2.221
Detector
TypeIDDetectorDate
DECTRIS EIGER X 16M1PIXELFeb 27, 2021
DECTRIS PILATUS3 2M2PIXELJun 11, 2021
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1Si(111)SINGLE WAVELENGTHMx-ray1
2Si(111)SINGLE WAVELENGTHMx-ray2
Radiation wavelength
IDWavelength (Å)Relative weight
111
21
ReflectionResolution: 2.34→29.44 Å / Num. obs: 89100 / % possible obs: 97.66 % / Redundancy: 1.7 % / Biso Wilson estimate: 39.81 Å2 / CC1/2: 0.948 / CC star: 0.987 / Rmerge(I) obs: 0.1977 / Rrim(I) all: 0.2728 / Net I/σ(I): 2.9
Reflection shellResolution: 2.34→2.4 Å / Redundancy: 1.7 % / Rmerge(I) obs: 0.7477 / Mean I/σ(I) obs: 0.88 / Num. unique obs: 9129 / CC1/2: 0.515 / CC star: 0.825 / % possible all: 79.5

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Processing

Software
NameVersionClassification
PHENIX1.21_5207refinement
PHENIX1.2phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.34→29.44 Å / SU ML: 0.3931 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.1712
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2769 2007 2.31 %
Rwork0.2394 84956 -
obs0.2403 86963 97.66 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 44.14 Å2
Refinement stepCycle: LAST / Resolution: 2.34→29.44 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14688 0 323 360 15371
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009615310
X-RAY DIFFRACTIONf_angle_d1.512520784
X-RAY DIFFRACTIONf_chiral_restr0.0852316
X-RAY DIFFRACTIONf_plane_restr0.0132676
X-RAY DIFFRACTIONf_dihedral_angle_d16.10115826
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAX-RAY DIFFRACTIONTorsion NCS0.543842311416
ens_1d_3AAX-RAY DIFFRACTIONTorsion NCS0.71852471511
ens_1d_4AAX-RAY DIFFRACTIONTorsion NCS0.607192779891
ens_1d_5AAX-RAY DIFFRACTIONTorsion NCS0.642352733117
ens_1d_6AAX-RAY DIFFRACTIONTorsion NCS1.49390222986
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.34-2.40.32331170.30434933X-RAY DIFFRACTION79.49
2.4-2.470.3471440.28756181X-RAY DIFFRACTION99.92
2.47-2.540.28541440.27446171X-RAY DIFFRACTION99.87
2.54-2.620.34851450.27166176X-RAY DIFFRACTION99.68
2.62-2.710.34191500.27576246X-RAY DIFFRACTION99.95
2.71-2.820.34551440.26696150X-RAY DIFFRACTION99.68
2.82-2.950.33921490.25826193X-RAY DIFFRACTION99.86
2.95-3.110.30711450.25256141X-RAY DIFFRACTION99.41
3.11-3.30.29591500.2496198X-RAY DIFFRACTION99.22
3.3-3.550.26491400.23076068X-RAY DIFFRACTION98.23
3.55-3.910.22911450.22176088X-RAY DIFFRACTION97.99
3.91-4.480.21421450.20446168X-RAY DIFFRACTION98.78
4.48-5.630.26231420.21846144X-RAY DIFFRACTION98.26
5.63-29.440.27241470.2416099X-RAY DIFFRACTION96.93
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.398266704947-0.1956097876830.1775470967740.726552245877-0.4093959806931.33112922844-0.0539291875263-0.0747292136081-0.1955335657920.148384028654-0.0006511526181020.01242463139010.156861390248-0.1344274898980.0530155249640.342674429324-0.01412410185010.04894205068060.372466633529-0.006179176549010.31511915358-24.5084173448-48.14217405821.54443436211
2-0.09540287473210.211874507730.05510204817230.189325204182-0.2537330518081.050801124360.006516030843150.1458894043230.106632767707-0.1227856116050.0217042529465-0.00352542395693-0.111754385516-0.102894044005-0.02997810333060.4101838861240.003437596024220.1017209247750.4687255910320.01285705598440.39461478253-24.4943430509-15.9899063786-25.690447264
32.158222536220.259219714591-1.056272921240.6761082588370.05667126957351.01924162393-0.0571016025865-0.210670688534-0.216870184460.0480259316573-0.04258356507210.03955453434610.1239776114790.01115887711730.1055588911720.319906569920.006009581071750.01260471188080.4407983772260.02456481672470.31028741296512.8272165095-53.7163872122-25.951401023
41.547317635440.688764878566-0.5033037669051.56857875698-0.2427557160440.663241552390.03005094107940.005674025910390.0663733248642-0.130111336499-0.0853160535088-0.3092781786310.03912663523370.1172512617950.0613105921880.3418126883420.04107089074470.06499885720480.4460445247390.04840472379610.34870614482843.9134865899-50.958522234-53.9771093574
51.58141846847-0.4892131313330.464957036960.969540341561-0.1128999382420.630230085634-0.0774899171052-0.1868751832380.05256055324820.1818405685260.0147338032131-0.114777945421-0.0364480776066-0.06405845182630.06217444108110.3839919734910.004940007204610.0526719226240.414580516532-0.03904331320020.3139488651294.34123275599-13.662923273-56.5314460081
61.97499991022-0.41881069431.62189479367-0.0340913142339-0.5003042362610.926625444332-0.0763529212498-0.0599882333480.16622805412-0.02231687186030.007537413629410.0229999799934-0.0859768951777-0.0688265259090.07817413234570.351077126839-0.005592730937320.08038223729680.427206940128-0.05400583638930.357624341865-27.0129037431-10.3489495517-84.635814602
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth seq-ID: 3 - 402 / Label seq-ID: 1

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-ID
11Chain AAA - C
22Chain BBD - F
33Chain CCG - I
44Chain DDJ - L
55Chain EEM - O
66Chain FFP - R

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