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- PDB-4ixt: Structure of a 37-fold mutant of halohydrin dehalogenase (HheC) b... -

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Basic information

Entry
Database: PDB / ID: 4ixt
TitleStructure of a 37-fold mutant of halohydrin dehalogenase (HheC) bound to ethyl (R)-4-cyano-3-hydroxybutyrate
ComponentsHalohydrin dehalogenase
KeywordsLYASE / thermostability / synergistic mutations / coupled mutations / proline-induced / backbone changes / enantioselectivity changes / directed evolution / protein engineering / short-chain dehydrogenase/reductase enzyme superfamily / Cyanolysis / dehalogenase / Atorvastatin synthesis
Function / homology
Function and homology information


: / Enoyl-(Acyl carrier protein) reductase / Short-chain dehydrogenase/reductase SDR / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ethyl (3R)-4-cyano-3-hydroxybutanoate / Halohydrin dehalogenase
Similarity search - Component
Biological speciesRhizobium radiobacter (Agrobacterium genomosp. 4)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.49 Å
AuthorsFloor, R.J. / Schallmey, M. / Hauer, B. / Breuer, M. / Jekel, P.A. / Wijma, H.J. / Dijkstra, B.W. / Janssen, D.B.
CitationJournal: Chembiochem / Year: 2013
Title: Biocatalytic and structural properties of a highly engineered halohydrin dehalogenase.
Authors: Schallmey, M. / Floor, R.J. / Hauer, B. / Breuer, M. / Jekel, P.A. / Wijma, H.J. / Dijkstra, B.W. / Janssen, D.B.
History
DepositionJan 28, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2013Provider: repository / Type: Initial release
Revision 1.1May 15, 2013Group: Database references
Revision 1.2Jan 24, 2018Group: Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.3Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / 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 / _struct_ref_seq_dif.details / _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

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Assembly

Deposited unit
A: Halohydrin dehalogenase
B: Halohydrin dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,8754
Polymers55,6832
Non-polymers1932
Water97354
1
A: Halohydrin dehalogenase
B: Halohydrin dehalogenase
hetero molecules

A: Halohydrin dehalogenase
B: Halohydrin dehalogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,7518
Polymers111,3664
Non-polymers3854
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_665-y+1,-x+1,-z+1/21
Buried area16080 Å2
ΔGint-129 kcal/mol
Surface area33150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.682, 104.682, 121.191
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Halohydrin dehalogenase


Mass: 27841.406 Da / Num. of mol.: 2 / Fragment: Halohydrin dehalogenase HheC
Mutation: Q37H, K38Q, K52I, A60V, Y70L, Q72H, V75I, F82A, A83P, P84V, F86W, Q87R, G99D, A100M, R107K, V112A, K121R, T134A, P135S, T146A, C153S, T154A, Y166H, G174A, Y177G, L178V, H179D, E181G, F186Y, ...Mutation: Q37H, K38Q, K52I, A60V, Y70L, Q72H, V75I, F82A, A83P, P84V, F86W, Q87R, G99D, A100M, R107K, V112A, K121R, T134A, P135S, T146A, C153S, T154A, Y166H, G174A, Y177G, L178V, H179D, E181G, F186Y, T189S, N195S, V201W, K203R, V205Y, A222T, M245V, I246V
Source method: isolated from a genetically manipulated source
Details: This enzyme is a result of a directed evolution study. It contains 37 mutations, compared to its parent
Source: (gene. exp.) Rhizobium radiobacter (Agrobacterium genomosp. 4)
Gene: hheC / Plasmid: pBAD-HheC-2360 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: Q93D82, Lyases; Carbon-halide lyases
#2: Chemical ChemComp-1H1 / ethyl (3R)-4-cyano-3-hydroxybutanoate


Mass: 157.167 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H11NO3
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 54 / 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: 2.98 Å3/Da / Density % sol: 58.74 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 12.5% (w/v) PEG 8000,0.2 M Magnesium Chloride, 0.1 M Tris-Cl pH 8.5, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-4 / Wavelength: 0.9395 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jul 20, 2010
RadiationMonochromator: Double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9395 Å / Relative weight: 1
ReflectionResolution: 2.47→73.36 Å / Num. obs: 27496 / % possible obs: 100 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 7.3 / Redundancy: 5.9 % / Rmerge(I) obs: 0.091 / Net I/σ(I): 14.3

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
PHASESphasing
REFMAC5.5.0070refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1PWZ

1pwz


Resolution: 2.49→52.34 Å / Cor.coef. Fo:Fc: 0.932 / Cor.coef. Fo:Fc free: 0.892 / SU B: 7.415 / SU ML: 0.173 / Cross valid method: THROUGHOUT / σ(F): 2 / σ(I): 2 / ESU R: 0.364 / ESU R Free: 0.259 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24895 1233 5.1 %RANDOM
Rwork0.19945 ---
all0.26 22989 --
obs0.20192 22989 99.93 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 29.067 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å2-0 Å20 Å2
2---0.05 Å2-0 Å2
3---0.09 Å2
Refinement stepCycle: LAST / Resolution: 2.49→52.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3811 0 12 54 3877
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0223924
X-RAY DIFFRACTIONr_angle_refined_deg1.1151.9485344
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.1365492
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.39424.142169
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.20615606
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.9941517
X-RAY DIFFRACTIONr_chiral_restr0.0930.2587
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.0213011
X-RAY DIFFRACTIONr_mcbond_it1.2861.52464
X-RAY DIFFRACTIONr_mcangle_it2.30423958
X-RAY DIFFRACTIONr_scbond_it3.78531460
X-RAY DIFFRACTIONr_scangle_it6.0064.51386
LS refinement shellResolution: 2.49→2.553 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.292 77 -
Rwork0.242 1682 -
obs-3471 100 %

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