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- PDB-1gk0: Structure-based prediction of modifications in glutarylamidase to... -

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Basic information

Entry
Database: PDB / ID: 1gk0
TitleStructure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C
Components(CEPHALOSPORIN ACYLASE) x 2
KeywordsHYDROLASE / CEPHALOSPORIN ACYLASE / GLUTARYL ACYLASE / CEPHALOSPORIN C / CATALYTIC TRIAD / NTN-HYDROLASE / X-RAZ STRUCTURE
Function / homology
Function and homology information


glutaryl-7-aminocephalosporanic-acid acylase / glutaryl-7-aminocephalosporanic-acid acylase activity / hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides / antibiotic biosynthetic process / periplasmic space / response to antibiotic / metal ion binding
Similarity search - Function
Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin/GL-7-ACA/AHL acylase / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL/aculeacin-A acylase / Penicillin amidase type, domain 1 / Penicillin amidase type, N-terminal domain, B-knob ...Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin/GL-7-ACA/AHL acylase / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL/aculeacin-A acylase / Penicillin amidase type, domain 1 / Penicillin amidase type, N-terminal domain, B-knob / Penicillin amidase type, A-knob / Penicillin amidase / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Roll / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / Cephalosporin acylase / Glutaryl-7-aminocephalosporanic-acid acylase
Similarity search - Component
Biological speciesPSEUDOMONAS SP. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.5 Å
AuthorsFritz-Wolf, K. / Koller, K.P. / Lange, G. / Liesum, A. / Sauber, K. / Schreuder, H. / Aretz, W. / Kabsch, W.
CitationJournal: Protein Sci. / Year: 2002
Title: Structure-Based Prediction of Modifications in Glutarylamidase to Allow Single-Step Enzymatic Production of 7-Aminocephalosporanic Acid from Cephalosporin C.
Authors: Fritz-Wolf, K. / Koller, K. / Lange, G. / Liesum, A. / Sauber, K. / Schreuder, H. / Aretz, W. / Kabsch, W.
History
DepositionAug 7, 2001Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2002Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3May 8, 2019Group: Data collection / Derived calculations ...Data collection / Derived calculations / Experimental preparation / Other
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow / pdbx_database_proc / pdbx_database_status / pdbx_seq_map_depositor_info / struct_conn
Item: _exptl_crystal_grow.method / _exptl_crystal_grow.temp ..._exptl_crystal_grow.method / _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval / _pdbx_seq_map_depositor_info.one_letter_code_mod / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Nov 6, 2024Group: Data collection / Database references ...Data collection / Database references / Other / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _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: CEPHALOSPORIN ACYLASE
B: CEPHALOSPORIN ACYLASE
C: CEPHALOSPORIN ACYLASE
D: CEPHALOSPORIN ACYLASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,0038
Polymers151,6894
Non-polymers3144
Water12,683704
1
A: CEPHALOSPORIN ACYLASE
B: CEPHALOSPORIN ACYLASE
hetero molecules

A: CEPHALOSPORIN ACYLASE
B: CEPHALOSPORIN ACYLASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,0038
Polymers151,6894
Non-polymers3144
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_656-x+1,y,-z+11
Buried area18500 Å2
ΔGint-142.1 kcal/mol
Surface area60880 Å2
MethodPQS
2
C: CEPHALOSPORIN ACYLASE
D: CEPHALOSPORIN ACYLASE
hetero molecules

C: CEPHALOSPORIN ACYLASE
D: CEPHALOSPORIN ACYLASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,0038
Polymers151,6894
Non-polymers3144
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area20740 Å2
ΔGint-148.2 kcal/mol
Surface area57990 Å2
MethodPQS
Unit cell
Length a, b, c (Å)228.366, 69.910, 113.550
Angle α, β, γ (deg.)90.00, 97.57, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein CEPHALOSPORIN ACYLASE / GLUTARYLAMIDASE


Mass: 16988.514 Da / Num. of mol.: 2 / Fragment: RESIDUES A8-A160
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PSEUDOMONAS SP. (bacteria) / Production host: ESCHERICHIA COLI (E. coli)
References: UniProt: O86089, UniProt: P07662*PLUS, penicillin amidase
#2: Protein CEPHALOSPORIN ACYLASE / GLUTARYLAMIDASE


Mass: 58855.785 Da / Num. of mol.: 2 / Fragment: RESIDUES B1-B522
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PSEUDOMONAS SP. (bacteria) / Production host: ESCHERICHIA COLI (E. coli)
References: UniProt: O86089, UniProt: P07662*PLUS, penicillin amidase
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 704 / Source method: isolated from a natural source / Formula: H2O
Compound detailsCATALYTICLLY ACTIVE CEPHALOSPORIN ACYLASE IS FORMED BY A TWO-STEP AUTOCATALYTIC PROCESS FROM THE ...CATALYTICLLY ACTIVE CEPHALOSPORIN ACYLASE IS FORMED BY A TWO-STEP AUTOCATALYTIC PROCESS FROM THE INACTIVE INTACT PRECURSOR. IN THE FIRST STEP THE PRECURSOR POLYPEPTIDE CHAIN IS CLEAVED INTO A SHORT (A1-A170) AND A LONG CHAIN (B1-B522). IN THE SECOND STEP THE SPACER PEPTIDE A161-A170 IS REMOVED. THE HETERO-DIMER IS NUMBERED HERE AS RESIDUES A8-A160 AND B1-B522.
Has protein modificationY

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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 Å3/Da / Density % sol: 58 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8
Details: CRYSTALS GROWN AT 18 C, HANGING DROP PRECIPITATION AGENT: 1.5-2.0 M POTASSIUM PHOSPHATE PH:7.0 -9.0, pH 8.00
Crystal grow
*PLUS
Temperature: 18 ℃ / pH: 7.5 / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
120-30 mg/mlprotein1drop
20.5 Mpotassium phosphate1droppH7.5
35 mMdithiothreitol1drop
41.5-2.0 potassium phosphate1reservoirpH7.0-9.0

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.8854,0.9776,0.9779
DetectorType: MAR scanner 345 mm plate / Detector: IMAGE PLATE
RadiationProtocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.88541
20.97761
30.97791
ReflectionResolution: 2.5→20 Å / Num. obs: 118686 / % possible obs: 96.8 % / Redundancy: 2.05 % / Biso Wilson estimate: 20.4 Å2 / Rmerge(I) obs: 0.045
Reflection shellResolution: 2.5→2.6 Å / Redundancy: 1.63 % / Rmerge(I) obs: 0.095 / % possible all: 79.4

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Processing

Software
NameVersionClassification
CNS1refinement
XDSdata reduction
XDSdata scaling
XSCALEdata scaling
CNSphasing
RefinementMethod to determine structure: MAD / Resolution: 2.5→19.48 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 8082389.02 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.222 3585 3 %RANDOM
Rwork0.207 ---
obs0.207 118686 98.7 %-
Solvent computationSolvent model: FLAT MODEL / ksol: 0.36922 e/Å3
Displacement parametersBiso mean: 14.1 Å2
Baniso -1Baniso -2Baniso -3
1--5.31 Å20 Å2-0.21 Å2
2--6.52 Å20 Å2
3----1.21 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.3 Å0.28 Å
Luzzati d res low-5 Å
Luzzati sigma a0.33 Å0.27 Å
Refinement stepCycle: LAST / Resolution: 2.5→19.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10650 0 18 704 11372
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d24.3
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.86
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it0.851.5
X-RAY DIFFRACTIONc_mcangle_it1.362
X-RAY DIFFRACTIONc_scbond_it1.552
X-RAY DIFFRACTIONc_scangle_it2.212.5
LS refinement shellResolution: 2.5→2.66 Å / Rfactor Rfree error: 0.012 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.289 539 2.8 %
Rwork0.256 18920 -
obs--97.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3ION.PARAMION.TOP
X-RAY DIFFRACTION4COL.PARCOL.TOP
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
Lowest resolution: 20 Å
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.3
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.86
LS refinement shell
*PLUS
Rfactor obs: 0.256

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