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- PDB-7s3v: Structure of HsKYNase_66, an evolved variant of human kynureninas... -

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Basic information

Entry
Database: PDB / ID: 7s3v
TitleStructure of HsKYNase_66, an evolved variant of human kynureninase with greatly increased activity towards kynurenine
ComponentsKynureninase
KeywordsANTITUMOR PROTEIN / HYDROLASE / Kynurenine / tryptophan / cancer / directed-evolution
Function / homology
Function and homology information


kynureninase / kynureninase activity / response to vitamin B6 / L-kynurenine catabolic process / anthranilate metabolic process / L-tryptophan catabolic process to kynurenine / quinolinate biosynthetic process / 'de novo' NAD+ biosynthetic process from L-tryptophan / L-tryptophan catabolic process / Tryptophan catabolism ...kynureninase / kynureninase activity / response to vitamin B6 / L-kynurenine catabolic process / anthranilate metabolic process / L-tryptophan catabolic process to kynurenine / quinolinate biosynthetic process / 'de novo' NAD+ biosynthetic process from L-tryptophan / L-tryptophan catabolic process / Tryptophan catabolism / NAD+ biosynthetic process / response to type II interferon / pyridoxal phosphate binding / protein homodimerization activity / mitochondrion / nucleoplasm / cytosol / cytoplasm
Similarity search - Function
Kynureninase / Kynureninase C-terminal domain / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.249 Å
AuthorsBurkholder, N.T. / Zhang, Y.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM104896 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125882 United States
CitationJournal: Nat Catal / Year: 2022
Title: Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.
Authors: Blazeck, J. / Karamitros, C.S. / Ford, K. / Somody, C. / Qerqez, A. / Murray, K. / Burkholder, N.T. / Marshall, N. / Sivakumar, A. / Lu, W.C. / Tan, B. / Lamb, C. / Tanno, Y. / Siddiqui, M.Y. ...Authors: Blazeck, J. / Karamitros, C.S. / Ford, K. / Somody, C. / Qerqez, A. / Murray, K. / Burkholder, N.T. / Marshall, N. / Sivakumar, A. / Lu, W.C. / Tan, B. / Lamb, C. / Tanno, Y. / Siddiqui, M.Y. / Ashoura, N. / Coma, S. / Zhang, X.M. / McGovern, K. / Kumada, Y. / Zhang, Y.J. / Manfredi, M. / Johnson, K.A. / D'Arcy, S. / Stone, E. / Georgiou, G.
History
DepositionSep 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Kynureninase
B: Kynureninase


Theoretical massNumber of molelcules
Total (without water)106,0202
Polymers106,0202
Non-polymers00
Water00
1
A: Kynureninase

A: Kynureninase


Theoretical massNumber of molelcules
Total (without water)106,0202
Polymers106,0202
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_454-x-1/2,y,-z-1/41
Buried area6530 Å2
ΔGint-57 kcal/mol
Surface area31730 Å2
MethodPISA
2
B: Kynureninase

B: Kynureninase


Theoretical massNumber of molelcules
Total (without water)106,0202
Polymers106,0202
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation16_444-y-1/2,-x-1/2,-z-1/21
Buried area6490 Å2
ΔGint-57 kcal/mol
Surface area32020 Å2
MethodPISA
Unit cell
Length a, b, c (Å)140.890, 140.890, 286.371
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number98
Space group name H-MI4122

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Components

#1: Protein Kynureninase / L-kynurenine hydrolase


Mass: 53009.840 Da / Num. of mol.: 2
Mutation: N67D, L72N, A99I, H102W, E103F, V104E, K106D, R107S, T111H, G112Y, A132V, A136T, M189I, V223I, F225Y, S274G, A280S, G281S, A282P, I331C, N333T, S341I, I405L, S408N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KYNU / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q16719, kynureninase
Has ligand of interestY

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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: 3.35 Å3/Da / Density % sol: 63.3 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: 8% PEG8000, 100 mM imidazole, 50 mM MgCl2, 0.2 mM PLP, 5% sucrose (diffracting crystal proteins were briefly supplemented with 0.005 mg/mL trypsin prior to sitting drop vapor diffusion in ...Details: 8% PEG8000, 100 mM imidazole, 50 mM MgCl2, 0.2 mM PLP, 5% sucrose (diffracting crystal proteins were briefly supplemented with 0.005 mg/mL trypsin prior to sitting drop vapor diffusion in order to remove flexible terminal ends).

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.03322 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 15, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03322 Å / Relative weight: 1
ReflectionResolution: 3.249→50 Å / Num. obs: 21474 / % possible obs: 92.7 % / Redundancy: 3.6 % / Biso Wilson estimate: 72.99 Å2 / Rmerge(I) obs: 0.164 / Rpim(I) all: 0.095 / Rrim(I) all: 0.191 / Χ2: 0.994 / Net I/σ(I): 4.3
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.25-3.313.60.79110640.5740.460.9210.94294.7
3.31-3.373.60.71910900.6350.4150.8360.97294.9
3.37-3.433.60.62110780.6890.3560.720.99994.7
3.43-3.53.60.48710680.7860.2820.5660.99994.3
3.5-3.583.60.45310890.840.2610.5260.98894.7
3.58-3.663.50.40510530.8330.2390.4731.03494.6
3.66-3.753.40.34810710.8650.2090.4091.03593.5
3.75-3.853.40.27210630.9160.160.3181.0593.9
3.85-3.973.40.23310910.940.1420.2751.13194.5
3.97-4.093.60.20310840.9530.120.2371.10294
4.09-4.243.70.19610740.9520.1110.2261.07493.6
4.24-4.413.80.16310740.9640.0920.1881.04793.3
4.41-4.613.70.15210650.9660.0870.1761.06593.1
4.61-4.853.70.13310830.9730.0750.1531.07593
4.85-5.163.60.12810540.9760.0720.1480.98591.8
5.16-5.563.40.12610510.9730.0740.1471.00490.2
5.56-6.113.50.13210790.9710.0770.1540.94791.8
6.11-73.70.11210840.980.0620.1280.94991.2
7-8.813.70.07710800.990.0430.0890.86389.6
8.81-503.50.05610790.9920.0320.0650.61884.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASERphasing
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2HZP

2hzp


Resolution: 3.249→46.344 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / Phase error: 21.91 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2278 2000 9.74 %
Rwork0.1855 18541 -
obs0.1896 20541 88.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 138.87 Å2 / Biso mean: 61.7 Å2 / Biso min: 28.04 Å2
Refinement stepCycle: final / Resolution: 3.249→46.344 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7074 0 1174 0 8248
Biso mean--68.86 --
Num. residues----742
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0047256
X-RAY DIFFRACTIONf_angle_d0.7329868
X-RAY DIFFRACTIONf_chiral_restr0.0471104
X-RAY DIFFRACTIONf_plane_restr0.0051254
X-RAY DIFFRACTIONf_dihedral_angle_d3.5314290
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.2492-3.33050.31931030.284295866
3.3305-3.42050.31591280.2653119180
3.4205-3.52110.33421380.2491127287
3.5211-3.63470.27411430.2406133090
3.6347-3.76460.24691450.2206134192
3.7646-3.91520.25481510.1948139794
3.9152-4.09330.26721490.1845138794
4.0933-4.3090.21591490.1724138194
4.309-4.57870.20911500.164138993
4.5787-4.93190.16791480.1529137293
4.9319-5.42750.19911490.1624137991
5.4275-6.21130.21871480.1834137291
6.2113-7.81940.22621500.1852139191
7.8194-46.3440.18511490.1505138185

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