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- PDB-1hjf: Alteration of the co-substrate selectivity of deacetoxycephalospo... -

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Basic information

Entry
Database: PDB / ID: 1hjf
TitleAlteration of the co-substrate selectivity of deacetoxycephalosporin C synthase: The role of arginine-258
ComponentsDEACETOXYCEPHALOSPORIN C SYNTHASE
KeywordsOXIDOREDUCTASE / ALTERNATIVE 2-OXOACIDS / CEPHEM ANTIBIOTIC BIOSYNTHESIS / CHEMICAL COSUBSTRATE RESCUE / CO-SUBSTRATE SELECTIVITY / 2- OXOGLUTARATE-DEPENDENT OXYGENASE
Function / homology
Function and homology information


deacetoxycephalosporin-C synthase / deacetoxycephalosporin-C synthase activity / L-ascorbic acid binding / antibiotic biosynthetic process / iron ion binding
Similarity search - Function
Isopenicillin N synthase, conserved site / Isopenicillin N synthase signature 2. / B-lactam Antibiotic, Isopenicillin N Synthase; Chain / non-haem dioxygenase in morphine synthesis N-terminal / Isopenicillin N synthase-like, Fe(2+) 2OG dioxygenase domain / 2OG-Fe(II) oxygenase superfamily / Isopenicillin N synthase-like superfamily / Oxoglutarate/iron-dependent dioxygenase / Fe(2+) 2-oxoglutarate dioxygenase domain profile. / Jelly Rolls ...Isopenicillin N synthase, conserved site / Isopenicillin N synthase signature 2. / B-lactam Antibiotic, Isopenicillin N Synthase; Chain / non-haem dioxygenase in morphine synthesis N-terminal / Isopenicillin N synthase-like, Fe(2+) 2OG dioxygenase domain / 2OG-Fe(II) oxygenase superfamily / Isopenicillin N synthase-like superfamily / Oxoglutarate/iron-dependent dioxygenase / Fe(2+) 2-oxoglutarate dioxygenase domain profile. / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
2-OXO-4-METHYLPENTANOIC ACID / : / Deacetoxycephalosporin C synthase
Similarity search - Component
Biological speciesSTREPTOMYCES CLAVULIGERUS (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsLee, H.J. / Lloyd, M.D. / Clifton, I.J. / Harlos, K. / Dubus, A. / Baldwin, J.E. / Frere, J.M. / Schofield, C.J.
Citation
Journal: J.Biol.Chem. / Year: 2001
Title: Alteration of the 2-Oxoacid Cosubstrate Selectivity in Deacetoxycephalosporin C Synthase: The Role of Arginine-258
Authors: Lee, H.J. / Lloyd, M.D. / Harlos, K. / Clifton, I.J. / Baldwin, J.E. / Schofield, C.J.
#1: Journal: J.Mol.Biol. / Year: 1999
Title: Studies on the Active Site of Deacetoxycephalosporin C Synthase
Authors: Lloyd, M.D. / Lee, H.J. / Harlos, K. / Zhang, Z.H. / Baldwin, J.E. / Schofield, C.J. / Charnock, J.M. / Garner, C.D. / Hara, T. / Van Scheltinga, A.C.T. / Valegard, K. / Viklund, J.A.C. / ...Authors: Lloyd, M.D. / Lee, H.J. / Harlos, K. / Zhang, Z.H. / Baldwin, J.E. / Schofield, C.J. / Charnock, J.M. / Garner, C.D. / Hara, T. / Van Scheltinga, A.C.T. / Valegard, K. / Viklund, J.A.C. / Hajdu, J. / Andersson, I. / Danielsson, A. / Bhikhabhai, R.
History
DepositionJan 15, 2001Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 1, 2001Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2013Group: Atomic model / Non-polymer description ...Atomic model / Non-polymer description / Other / Version format compliance
Revision 1.2Oct 24, 2018Group: Data collection / Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain
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 / pdbx_struct_conn_angle / struct_conn / 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 / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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 / _struct_conn.ptnr2_label_seq_id / _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: DEACETOXYCEPHALOSPORIN C SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,7493
Polymers34,5631
Non-polymers1862
Water1,78399
1
A: DEACETOXYCEPHALOSPORIN C SYNTHASE
hetero molecules

A: DEACETOXYCEPHALOSPORIN C SYNTHASE
hetero molecules

A: DEACETOXYCEPHALOSPORIN C SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,2469
Polymers103,6883
Non-polymers5586
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
MethodPQS
Unit cell
Length a, b, c (Å)106.933, 106.933, 70.819
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3

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Components

#1: Protein DEACETOXYCEPHALOSPORIN C SYNTHASE / DAOCS


Mass: 34562.574 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: IRON(II) AND ALPHA-KETO-ISOCAPROATE COMPLEX OF MUTANT ENZYME
Source: (gene. exp.) STREPTOMYCES CLAVULIGERUS (bacteria)
Description: RECOMBINANT ENZYME OF S. CLAVULIGERUS ENZYME. R258Q MUTANT ENZYME
Gene: CEFE, R258Q MUTANT / Plasmid: PET24A / Production host: ESCHERICHIA COLI BL21(DE3) (bacteria) / References: UniProt: P18548
#2: Chemical ChemComp-FE2 / FE (II) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#3: Chemical ChemComp-COI / 2-OXO-4-METHYLPENTANOIC ACID / alpha-ketoisocaproic acid / Alpha-Ketoisocaproic acid


Mass: 130.142 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H10O3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 99 / Source method: isolated from a natural source / Formula: H2O
Compound detailsCHAIN A ENGINEERED MUTATION ARG258GLN DAOCS CATALYZES THE REACTION FROM PENICILLIN N TO DEACETOXY- ...CHAIN A ENGINEERED MUTATION ARG258GLN DAOCS CATALYZES THE REACTION FROM PENICILLIN N TO DEACETOXY- CEPHALOSPORIN C.
Sequence detailsRESIDUE A50 BUILT AS ILE (AS REPORTED IN PATENT) NOT LEU (AS REPORTED IN PAPER) SIDECHAIN OF GLN- ...RESIDUE A50 BUILT AS ILE (AS REPORTED IN PATENT) NOT LEU (AS REPORTED IN PAPER) SIDECHAIN OF GLN-258 DISORDERED.

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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: 2.42 Å3/Da / Density % sol: 48.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: HANGING DROP AT 20 DEGREES C. 100 MM HEPES-NAOH, PH 7.0, 6% (W/V) GLYCEROL, 5 MM 2-OXO-4-METHYLPENTANOATE, 1.5-1.7 M AMMONIUM SULPHATE CRYSTALS SOAKED IN 5 MM IRON(II) SULPHATE IN MOTHER ...Details: HANGING DROP AT 20 DEGREES C. 100 MM HEPES-NAOH, PH 7.0, 6% (W/V) GLYCEROL, 5 MM 2-OXO-4-METHYLPENTANOATE, 1.5-1.7 M AMMONIUM SULPHATE CRYSTALS SOAKED IN 5 MM IRON(II) SULPHATE IN MOTHER LIQUOR UNDER ANAEROBIC CONDITIONS BEFORE DATA COLLECTION.
Crystal grow
*PLUS
Temperature: 14 ℃ / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
1100 mMNaOH/HEPES1reservoir
21.5-1.7 Mammonium sulfate1reservoir
33-6 %(w/v)glycerol1reservoir
45 mM2-oxo-glutarate1reservoir
55 mM2-oxo-4-methylpentanoate1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SRS / Beamline: PX9.6 / Wavelength: 0.87
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Jul 10, 1999
RadiationMonochromator: SILICON 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.87 Å / Relative weight: 1
ReflectionResolution: 1.6→100 Å / Num. obs: 32966 / % possible obs: 95.7 % / Observed criterion σ(I): 1 / Redundancy: 3.3 % / Biso Wilson estimate: 28.3 Å2 / Rmerge(I) obs: 0.07 / Net I/σ(I): 7.5
Reflection shellResolution: 1.6→1.69 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.728 / Mean I/σ(I) obs: 1 / % possible all: 95.7
Reflection
*PLUS
Lowest resolution: 100 Å / Num. measured all: 122299 / Rmerge(I) obs: 0.07
Reflection shell
*PLUS
% possible obs: 97.5 %

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Processing

Software
NameVersionClassification
CNS0.9refinement
MOSFLMdata reduction
SCALAdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1RXG
Resolution: 1.6→19.38 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 1783912 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Details: DATA DETWINNED BEFORE REFINEMENT
RfactorNum. reflection% reflectionSelection details
Rfree0.269 1442 4.1 %RANDOM
Rwork0.241 ---
obs0.241 35225 88.3 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 61.9845 Å2 / ksol: 0.396472 e/Å3
Displacement parametersBiso mean: 30.3 Å2
Baniso -1Baniso -2Baniso -3
1--1.8 Å2-0.21 Å20 Å2
2---1.8 Å20 Å2
3---3.61 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.29 Å0.26 Å
Luzzati d res low-5 Å
Luzzati sigma a0.31 Å0.3 Å
Refinement stepCycle: LAST / Resolution: 1.6→19.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2147 0 10 99 2256
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.013
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_deg1.2
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.6
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.66
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
LS refinement shellResolution: 1.6→1.7 Å / Rfactor Rfree error: 0.028 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.389 193 3.8 %
Rwork0.404 4887 -
obs--76.5 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1CNS_TOPPAR:PROTEIN_REP.PARAMCNS_TOPPAR:PROTEIN_REP.TOP
X-RAY DIFFRACTION2CNS_TOPPAR:ION.PARAMCNS_TOPPAR:ION.TOP
X-RAY DIFFRACTION3CNS_TOPPAR:WATER_REP.PARAMCNS_TOPPAR:WATER_REP.TOP
X-RAY DIFFRACTION4AKIC.PAR, ION_PATCH.PARAKIC.TOP, IRON_PATCH.TOP
Software
*PLUS
Name: CNS / Version: 0.5 / Classification: refinement
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.66

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