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- PDB-2cla: CRYSTAL STRUCTURE OF THE ASP-199-ASN MUTANT OF CHLORAMPHENICOL AC... -

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Entry
Database: PDB / ID: 2cla
TitleCRYSTAL STRUCTURE OF THE ASP-199-ASN MUTANT OF CHLORAMPHENICOL ACETYLTRANSFERASE TO 2.35 ANGSTROMS RESOLUTION. STRUCTURAL CONSEQUENCES OF DISRUPTION OF A BURIED SALT-BRIDGE
ComponentsCHLORAMPHENICOL ACETYLTRANSFERASE
KeywordsTRANSFERASE (ACYLTRANSFERASE)
Function / homology
Function and homology information


chloramphenicol O-acetyltransferase / chloramphenicol O-acetyltransferase activity / response to antibiotic
Similarity search - Function
Chloramphenicol acetyltransferase, active site / Chloramphenicol acetyltransferase active site. / Chloramphenicol acetyltransferase / Chloramphenicol acetyltransferase / Chloramphenicol acetyltransferase / Chloramphenicol Acetyltransferase / Chloramphenicol acetyltransferase-like domain / Chloramphenicol acetyltransferase-like domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
: / Chloramphenicol acetyltransferase 3
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / Resolution: 2.35 Å
AuthorsGibbs, M.R. / Moody, P.C.E. / Leslie, A.G.W.
Citation
Journal: Biochemistry / Year: 1990
Title: Crystal structure of the aspartic acid-199----asparagine mutant of chloramphenicol acetyltransferase to 2.35-A resolution: structural consequences of disruption of a buried salt bridge.
Authors: Gibbs, M.R. / Moody, P.C. / Leslie, A.G.
#1: Journal: Biochemistry / Year: 1990
Title: Evidence for Transition-State Stabilization by Serine-148 in the Catalytic Mechanism of Chloramphenicol Acetyltransferase
Authors: Lewendon, A. / Murray, I.A. / Shaw, W.V. / Gibbs, M.R. / Leslie, A.G.W.
#2: Journal: J.Mol.Biol. / Year: 1990
Title: Refined Crystal Structure of Type III Chloramphenicol Acetyltransferase at 1.75 Angstroms Resolution
Authors: Leslie, A.G.W.
#3: Journal: Biochemistry / Year: 1988
Title: Substitutions in the Active Site of Chloramphenicol Acetyltransferase. Role of a Conserved Aspartate
Authors: Lewendon, A. / Murray, I.A. / Kleanthous, C. / Cullis, P.M. / Shaw, W.V.
#4: Journal: Proc.Natl.Acad.Sci.USA / Year: 1988
Title: Structure of Chloramphenicol Acetyltransferase at 1.75-Angstroms Resolution
Authors: Leslie, A.G.W. / Moody, P.C.E. / Shaw, W.V.
#5: Journal: J.Mol.Biol. / Year: 1986
Title: Crystallization of a Type III Chloramphenicol Acetyl Transferase
Authors: Leslie, A.G.W. / Liddell, J.M. / Shaw, W.V.
History
DepositionApr 5, 1990Processing site: BNL
Revision 1.0Jul 15, 1990Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Feb 14, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_struct_conn_angle / pdbx_struct_special_symmetry / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.process_site / _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.ptnr1_symmetry / _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.ptnr3_symmetry / _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.ptnr1_symmetry / _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_conn.ptnr2_symmetry / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700SHEET SHEET 1 ACTUALLY HAS SEVEN STRANDS. DATA BANK CONVENTIONS DO NOT ALLOW LISTING RESIDUES WHICH ...SHEET SHEET 1 ACTUALLY HAS SEVEN STRANDS. DATA BANK CONVENTIONS DO NOT ALLOW LISTING RESIDUES WHICH ARE SYMMETRY RELATED. THE FINAL STRAND, SER 157 - VAL 162, IS IN AN ADJACENT (THREE-FOLD RELATED) SUBUNIT OF THE TRIMER. N OF ASN 159 IS HYDROGEN BONDED TO O OF SEH 34. THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177, TYR 178, AND LEU 187. RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX STRANDED BETA-SHEET OF AN ADJACENT SHEET WHICH SPANS THE SUBUNIT INTERFACE.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,1383
Polymers25,0211
Non-polymers1182
Water1,874104
1
A: CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules

A: CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules

A: CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,4159
Polymers75,0623
Non-polymers3546
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area6410 Å2
ΔGint-34 kcal/mol
Surface area25390 Å2
MethodPISA, PQS
2
A: CHLORAMPHENICOL ACETYLTRANSFERASE
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)150,83018
Polymers150,1236
Non-polymers70712
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
crystal symmetry operation4_555y,x,-z1
crystal symmetry operation5_555x-y,-y,-z1
crystal symmetry operation6_555-x,-x+y,-z1
Buried area14150 Å2
ΔGint-119 kcal/mol
Surface area49820 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.700, 107.700, 124.400
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32
Atom site foot note1: THERE IS A WIDE BETA-BULGE INVOLVING RESIDUES LYS 177, TYR 178, AND LEU 187.
2: RESIDUES 157 - 162 FORM AN EXTENSION TO THE SIX STRANDED BETA-SHEET OF AN ADJACENT SHEET WHICH SPANS THE SUBUNIT INTERFACE.
Components on special symmetry positions
IDModelComponents
11A-222-

CO

21A-223-

CO

31A-310-

HOH

41A-311-

HOH

51A-491-

HOH

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Components

#1: Protein CHLORAMPHENICOL ACETYLTRANSFERASE /


Mass: 25020.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
References: UniProt: P00484, chloramphenicol O-acetyltransferase
#2: Chemical ChemComp-CO / COBALT (II) ION


Mass: 58.933 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Co
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 104 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE MUTATED ASPARTATE RESIDUE IS BELIEVED TO HAVE A STRUCTURAL ROLE AND IS SITUATED NEAR THE ACTIVE ...THE MUTATED ASPARTATE RESIDUE IS BELIEVED TO HAVE A STRUCTURAL ROLE AND IS SITUATED NEAR THE ACTIVE SITE. (SEE REFERENCE 3 ABOVE). THE ASP 199 TO ASN MUTANT SHOWS LARGE LOSS IN ACTIVITY (K=CAT= REDUCED 1500-FOLD) BUT WITH NEAR WILD TYPE THERMOSTABILITY. THIS MUTATION CAUSES ACTIVE SITE CONFORMATIONAL CHANGES INCLUDING THE CATALYTICALLY ESSENTIAL HISTIDINE, AND ALSO CAUSES CHANGES UP TO 20 ANGSTROMS AWAY.
Sequence detailsTHE NUMBERING SCHEME ADOPTED IS BASED ON THE ALIGNMENT OF A NUMBER OF CAT SEQUENCES. FOR THE TYPE ...THE NUMBERING SCHEME ADOPTED IS BASED ON THE ALIGNMENT OF A NUMBER OF CAT SEQUENCES. FOR THE TYPE III ENZYME WHOSE COORDINATES ARE PRESENTED IN THIS ENTRY, MET 6 IS THE N-TERMINAL RESIDUE AND THERE IS NO RESIDUE NUMBER 79.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.77 Å3/Da / Density % sol: 55.65 %
Crystal grow
*PLUS
Temperature: 4 ℃ / pH: 6.3 / Method: microdialysis
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
15 mg/mlprotein11
210 mMMES11
34 %MPD12
410 mMMES12
51 mMCm12
60.5 mMhexaamminecobalt(III)chloride12
75 mMbeta-mercaptoethanol12

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Data collection

Reflection
*PLUS
Highest resolution: 2.35 Å / % possible obs: 75 % / Rmerge(I) obs: 0.078

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Processing

SoftwareName: PROLSQ / Classification: refinement
RefinementRfactor obs: 0.152 / Highest resolution: 2.35 Å
Details: THE STRUCTURE WAS SOLVED BY MOLECULAR REPLACEMENT USING THE REFINED 1.75 ANGSTROMS RESOLUTION STRUCTURE OF THE WILD TYPE ENZYME AS A MODEL.
Refinement stepCycle: LAST / Highest resolution: 2.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1644 0 2 104 1750
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.020.02
X-RAY DIFFRACTIONp_angle_d0.040.03
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d0.060.05
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it
X-RAY DIFFRACTIONp_mcangle_it
X-RAY DIFFRACTIONp_scbond_it
X-RAY DIFFRACTIONp_scangle_it
X-RAY DIFFRACTIONp_plane_restr0.020.02
X-RAY DIFFRACTIONp_chiral_restr0.180.15
X-RAY DIFFRACTIONp_singtor_nbd0.160.2
X-RAY DIFFRACTIONp_multtor_nbd0.180.2
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
Refinement
*PLUS
Highest resolution: 2.35 Å / Rfactor obs: 0.152
Solvent computation
*PLUS
Displacement parameters
*PLUS

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