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- PDB-1gk1: Structure-based prediction of modifications in glutarylamidase to... -

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Basic information

Entry
Database: PDB / ID: 1gk1
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
Cephalosporin acylase / Glutaryl-7-aminocephalosporanic-acid acylase
Similarity search - Component
Biological speciesPSEUDOMONAS SP. (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.4 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.P. / 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 1.1Sep 7, 2011Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Non-polymer description / Other / Refinement description / Structure summary / Version format compliance
Revision 1.2Jun 28, 2017Group: Data collection / Category: diffrn_source / Item: _diffrn_source.type
Revision 1.3May 8, 2019Group: Data collection / Experimental preparation
Category: database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow
Item: _exptl_crystal_grow.method / _exptl_crystal_grow.temp
Revision 1.4Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
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 / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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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)150,7476
Polymers150,5634
Non-polymers1842
Water11,872659
1
A: CEPHALOSPORIN ACYLASE
B: CEPHALOSPORIN ACYLASE
hetero molecules

A: CEPHALOSPORIN ACYLASE
B: CEPHALOSPORIN ACYLASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,7476
Polymers150,5634
Non-polymers1842
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_656-x+1,y,-z+11
Buried area18450 Å2
ΔGint-142.6 kcal/mol
Surface area61270 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)150,7476
Polymers150,5634
Non-polymers1842
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area20670 Å2
ΔGint-152.1 kcal/mol
Surface area58890 Å2
MethodPQS
Unit cell
Length a, b, c (Å)230.290, 70.440, 114.800
Angle α, β, γ (deg.)90.00, 97.48, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein CEPHALOSPORIN ACYLASE / GLUTARYLAMIDASE


Mass: 16941.619 Da / Num. of mol.: 2 / Fragment: RESIDUES 36-188
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: 58339.941 Da / Num. of mol.: 2 / Fragment: RESIDUES 199-720
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-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 659 / 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.

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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
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: ROTATING ANODE / Type: ENRAF-NONIUS FR571 / Wavelength: 1.5418
DetectorType: MAR scanner 300 mm plate / Detector: IMAGE PLATE / Date: Feb 15, 1997
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.4→14.63 Å / Num. obs: 67222 / % possible obs: 94.1 % / Redundancy: 1.9 % / Biso Wilson estimate: 16.7 Å2 / Rmerge(I) obs: 0.09
Reflection shellResolution: 2.4→2.5 Å / Redundancy: 1.9 % / Rmerge(I) obs: 0.262 / % possible all: 92.7
Reflection
*PLUS
Lowest resolution: 15 Å / % possible obs: 93.7 % / Rmerge(I) obs: 0.093
Reflection shell
*PLUS
% possible obs: 92.7 %

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Processing

Software
NameVersionClassification
CNS1refinement
XDSdata reduction
XDSdata scaling
XSCALEdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1GK0

1gk0


Resolution: 2.4→14.63 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 6897765.46 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.219 3362 5 %RANDOM
Rwork0.18 ---
obs0.18 67222 94.1 %-
Solvent computationSolvent model: FLAT MODEL / ksol: 0.33435 e/Å3
Displacement parametersBiso mean: 19.6 Å2
Baniso -1Baniso -2Baniso -3
1--0.96 Å20 Å20.66 Å2
2--0.88 Å20 Å2
3---0.08 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.3 Å0.24 Å
Luzzati d res low-5 Å
Luzzati sigma a0.34 Å0.29 Å
Refinement stepCycle: LAST / Resolution: 2.4→14.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10650 0 12 659 11321
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.006
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_deg1.3
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d24
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.8
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.121.5
X-RAY DIFFRACTIONc_mcangle_it1.822
X-RAY DIFFRACTIONc_scbond_it2.022
X-RAY DIFFRACTIONc_scangle_it3.042.5
LS refinement shellResolution: 2.4→2.55 Å / Rfactor Rfree error: 0.012 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.281 549 5 %
Rwork0.247 10425 -
obs--92.8 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3ION.PARAMGOL.TOP
X-RAY DIFFRACTION4GOL.PAR
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
Lowest resolution: 15 Å / Rfactor obs: 0.18 / Rfactor Rwork: 0.18
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
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.8

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