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- PDB-1gkf: Crystal structures of penicillin acylase enzyme-substrate complex... -

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Basic information

Entry
Database: PDB / ID: 1gkf
TitleCrystal structures of penicillin acylase enzyme-substrate complexes: Structural insights into the catalytic mechanism
Components
  • PENICILLIN G ACYLASE ALPHA SUBUNIT
  • PENICILLIN G ACYLASE BETA SUBUNIT
KeywordsHYDROLASE / ANTIBIOTIC RESISTANCE
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 amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL acylase / Penicillin/GL-7-ACA/AHL/aculeacin-A acylase ...Helix Hairpins - #150 / Penicillin G acylase, C-terminal / Penicillin amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL acylase / 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 / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.41 Å
AuthorsMcVey, C.E. / Walsh, M.A. / Dodson, G.G. / Wilson, K.S. / Brannigan, J.A.
Citation
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.
#1: Journal: Protein Eng. / Year: 1990
Title: Expression, Purification and Crystallisation of Penicillin G Acylase from Escherichia Coli Atcc 11105
Authors: Hunt, P.D. / Tolley, S.P. / Ward, R.J. / Hill, C.P. / Dodson, G.G.
#2: 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.
History
DepositionAug 13, 2001Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 4, 2002Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2016Group: Atomic model / Database references ...Atomic model / Database references / Derived calculations / Non-polymer description / Other / Structure summary / Version format compliance
Revision 1.2Jul 24, 2019Group: Advisory / Data collection / Derived calculations
Category: diffrn_source / pdbx_unobs_or_zero_occ_atoms ...diffrn_source / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / struct_conn
Item: _diffrn_source.pdbx_synchrotron_site / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)92,21316
Polymers91,3662
Non-polymers84714
Water16,232901
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)51.200, 131.700, 63.900
Angle α, β, γ (deg.)90.00, 105.70, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 28979.695 Da / Num. of mol.: 1 / Fragment: N-TERMINAL NUCLEOPHILE DOMAIN RESIDUES 29-286
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Cellular location: PERIPLASM / Gene: PAC / Plasmid: PACYC184PAC / Cellular location (production host): PERIPLASM / Gene (production host): PAC / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P06875, penicillin amidase
#2: Protein PENICILLIN G ACYLASE BETA SUBUNIT


Mass: 62386.473 Da / Num. of mol.: 1 / Fragment: RESIDUES 287-846 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Cellular location: PERIPLASM / Gene: PAC / Plasmid: PACYC184PAC / Cellular location (production host): PERIPLASM / Gene (production host): PAC / 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.: 13 / 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.: 901 / Source method: isolated from a natural source / Formula: H2O
Compound detailsCHAIN B ENGINEERED MUTATION ASN530ALA

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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: 2.27 Å3/Da / Density % sol: 45.81 %
Crystal growpH: 7.5 / Details: pH 7.50
Crystal grow
*PLUS
Temperature: 291 K / pH: 7.2 / Method: vapor diffusion, hanging drop / Details: McVey, C.E., (1997) Acta Crystallog., D53, 777.
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
110 mg/mlprotein1drop
250 mMMOPS1droppH7.2
350 mMMOPS1reservoirpH7.2
410-12 %PEG2000 MME1reservoir
520-25 %ethylene glycol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: BW7B / Wavelength: 0.89
DetectorType: MAR scanner 300 mm plate / Detector: IMAGE PLATE
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.89 Å / Relative weight: 1
ReflectionResolution: 1.4→25 Å / Num. obs: 152163 / % possible obs: 97.4 % / Redundancy: 2.4 % / Biso Wilson estimate: 12.7 Å2 / Rmerge(I) obs: 0.052 / Net I/σ(I): 19.8
Reflection shellResolution: 1.41→1.43 Å / Rmerge(I) obs: 0.176 / Mean I/σ(I) obs: 4.4 / % possible all: 82.5
Reflection
*PLUS
Num. measured all: 361905
Reflection shell
*PLUS
% possible obs: 95.9 %

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Processing

Software
NameClassification
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.41→25 Å / SU B: 0.83 / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.168 4584 3 %RANDOM
Rwork0.148 ---
obs-147544 97.4 %-
Displacement parametersBiso mean: 14.4 Å2
Refinement stepCycle: LAST / Resolution: 1.41→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6075 0 53 901 7029
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.0180.02
X-RAY DIFFRACTIONp_angle_d0.0310.04
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d0.0380.05
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it1.5093
X-RAY DIFFRACTIONp_mcangle_it1.9875
X-RAY DIFFRACTIONp_scbond_it3.3957
X-RAY DIFFRACTIONp_scangle_it4.25310
X-RAY DIFFRACTIONp_plane_restr0.02550.03
X-RAY DIFFRACTIONp_chiral_restr0.1310.15
X-RAY DIFFRACTIONp_singtor_nbd
X-RAY DIFFRACTIONp_multtor_nbd
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd
X-RAY DIFFRACTIONp_planar_tor
X-RAY DIFFRACTIONp_staggered_tor
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor
X-RAY DIFFRACTIONp_special_tor
Software
*PLUS
Name: REFMAC / Classification: refinement
Refinement
*PLUS
Lowest resolution: 25 Å / Rfactor obs: 0.149 / Rfactor Rfree: 0.169
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_bond_d0.011
X-RAY DIFFRACTIONp_angle_d0.026
X-RAY DIFFRACTIONp_planar_d0.032

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