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- PDB-1h2g: Altered substrate specificity mutant of penicillin acylase -

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Basic information

Entry
Database: PDB / ID: 1h2g
TitleAltered substrate specificity mutant of penicillin acylase
Components
  • PENICILLIN G ACYLASE ALPHA SUBUNIT
  • PENICILLIN G ACYLASE BETA SUBUNIT
KeywordsHYDROLASE / AMIDOHYDROLASE / ANTIBIOTIC RESISTANCE / ALTERED SPECIFICITY / ZYMOGEN
Function / homology
Function and homology information


penicillin amidase activity / penicillin amidase / antibiotic biosynthetic process / periplasmic space / response to antibiotic / metal ion binding
Similarity search - Function
Helix Hairpins - #150 / Penicillin G acylase, C-terminal / 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 ...Helix Hairpins - #150 / Penicillin G acylase, C-terminal / 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 / Helix Hairpins / Roll / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Penicillin G acylase
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsMcVey, C.E. / Morillas, M. / Brannigan, J.A. / Ladurner, A.G. / Forney, L.J. / Virden, R.
Citation
Journal: Biochem.J. / Year: 2003
Title: Mutations of Penicillin Acylase Residue B71 Extend Substrate Specificity by Decreasing Steric Constraints for Substrate Binding
Authors: Morillas, M. / Mcvey, C.E. / Brannigan, J.A. / Ladurner, A.G. / Forney, L.J. / Virden, R.
#1: Journal: Nature / Year: 1995
Title: Penicillin Acylase Has a Single-Amino-Acid Catalytic Centre
Authors: Duggleby, H.J. / Tolley, S.P. / Hill, C.P. / Dodson, E.J. / Dodson, G. / Moody, P.C.E.
#2: Journal: J.Mol.Biol. / Year: 2001
Title: Crystal Structures of Penicillin Acylase Enzyme-Substrate Complexes: Structural Insights Into the Catalytic Mechanism
Authors: Mcvey, C.E. / Walsh, M.A. / Dodson, G.G. / Wilson, K.S. / Brannigan, J.A.
History
DepositionAug 8, 2002Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 17, 2003Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2014Group: Database references / Derived calculations ...Database references / Derived calculations / Non-polymer description / Other / Structure summary / Version format compliance
Revision 1.2Dec 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_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_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_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_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_asym_id / _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_asym_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: PENICILLIN G ACYLASE ALPHA SUBUNIT
B: PENICILLIN G ACYLASE BETA SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,5237
Polymers86,2342
Non-polymers2885
Water10,737596
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15930 Å2
ΔGint-116.3 kcal/mol
Surface area34630 Å2
MethodPQS
Unit cell
Length a, b, c (Å)52.020, 64.230, 70.670
Angle α, β, γ (deg.)70.58, 72.81, 73.84
Int Tables number1
Space group name H-MP1

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Components

#1: Protein PENICILLIN G ACYLASE ALPHA SUBUNIT / PENICILLIN AMIDOHYDROLASE / PENICILLIN G AMIDASE


Mass: 23838.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Plasmid: PACYC184PAC / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P06875, penicillin amidase
#2: Protein PENICILLIN G ACYLASE BETA SUBUNIT / PENICILLIN AMIDOHYDROLASE / PENICILLIN G AMIDASE


Mass: 62395.480 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: ENGINEERED MUTATION PHE 360 LEU / Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Plasmid: PACYC184PAC / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P06875, penicillin amidase
#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 596 / Source method: isolated from a natural source / Formula: H2O

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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: 2.4 Å3/Da / Density % sol: 48.9 %
Crystal growpH: 7.2 / Details: 50MM MOPS PH 7.2, 12% MME PEG2K, STREAK-SEEDING
Crystal grow
*PLUS
pH: 7.2 / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
111 mg/mlprotein1drop
211 %(w/v)PEG2000 MME1drop
350 mMMOPS1droppH7.2

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Data collection

DiffractionMean temperature: 120 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: RIGAKU IMAGE PLATE / Detector: IMAGE PLATE
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2→20 Å / Num. obs: 50677 / % possible obs: 93 % / Redundancy: 2 % / Rmerge(I) obs: 0.04 / Net I/σ(I): 14.8
Reflection shellResolution: 2→2.43 Å / Redundancy: 1.8 % / Rmerge(I) obs: 0.101 / Mean I/σ(I) obs: 7.1 / % possible all: 80
Reflection
*PLUS
Highest resolution: 2 Å / Lowest resolution: 19.8 Å / % possible obs: 93 % / Num. measured all: 102704 / Rmerge(I) obs: 0.04
Reflection shell
*PLUS
% possible obs: 80 % / Rmerge(I) obs: 0.101 / Mean I/σ(I) obs: 7.1

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Processing

Software
NameVersionClassification
REFMAC5.0.36refinement
DENZOdata reduction
Agrovatadata scaling
ROTAVATAdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1PNK

1pnk


Resolution: 2→19.8 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.935 / SU B: 10.31 / SU ML: 0.292 / Cross valid method: THROUGHOUT / ESU R: 0.173 / ESU R Free: 0.147 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.194 2536 5 %RANDOM
Rwork0.152 ---
obs-48141 100 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL PLUS MASK
Displacement parametersBiso mean: 16.47 Å2
Baniso -1Baniso -2Baniso -3
1--0.37 Å20.46 Å20.09 Å2
2---0.63 Å21.09 Å2
3----0.03 Å2
Refinement stepCycle: LAST / Resolution: 2→19.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6061 0 17 596 6674
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0216259
X-RAY DIFFRACTIONr_bond_other_d0.0010.025410
X-RAY DIFFRACTIONr_angle_refined_deg1.6831.9218527
X-RAY DIFFRACTIONr_angle_other_deg0.854312617
X-RAY DIFFRACTIONr_dihedral_angle_1_deg1.3683768
X-RAY DIFFRACTIONr_dihedral_angle_2_deg
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.99151065
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.1080.2898
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.027053
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021287
X-RAY DIFFRACTIONr_nbd_refined0.3260.31332
X-RAY DIFFRACTIONr_nbd_other0.2380.35328
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other1.1270.52
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1410.5522
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined0.080.54
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2090.38
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2940.323
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1490.511
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.09123804
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.836136
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.11222455
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it1.78832391
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2→2.05 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.214 128
Rwork0.164 2635
Refinement
*PLUS
Highest resolution: 2 Å / Lowest resolution: 19.8 Å / Num. reflection obs: 48181 / % reflection Rfree: 5 % / Rfactor Rfree: 0.0194 / Rfactor Rwork: 0.0152
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONr_bond_d0.013
X-RAY DIFFRACTIONr_angle_d
X-RAY DIFFRACTIONr_angle_deg1.7

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