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- PDB-5cge: Structure of Hydroxyethylthiazole Kinase ThiM from Staphylococcus... -

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Basic information

Entry
Database: PDB / ID: 5cge
TitleStructure of Hydroxyethylthiazole Kinase ThiM from Staphylococcus aureus in complex with substrate analog 2-(2-methyl-1H-imidazole-1-yl)ethanol
ComponentsHydroxyethylthiazole kinase
KeywordsTRANSFERASE / Bacterial Thiamine Biosynthesis / Hydroxyethylthiazole Kinase / Substrate Analog
Function / homology
Function and homology information


hydroxyethylthiazole kinase / hydroxyethylthiazole kinase activity / thiamine diphosphate biosynthetic process / thiamine biosynthetic process / magnesium ion binding / ATP binding
Similarity search - Function
Hydroxyethylthiazole kinase / Hydroxyethylthiazole kinase family / Ribokinase / Ribokinase-like / UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
2-(2-methyl-1H-imidazol-1-yl)ethanol / Hydroxyethylthiazole kinase
Similarity search - Component
Biological speciesStaphylococcus aureus subsp. aureus MRSA252 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 1.62 Å
AuthorsKuenz, M. / Drebes, J. / Windshuegel, B. / Cang, H. / Wrenger, C. / Betzel, C.
Funding support Germany, 1items
OrganizationGrant numberCountry
LEXI-SDI Germany
CitationJournal: Sci Rep / Year: 2016
Title: Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections.
Authors: Julia Drebes / Madeleine Künz / Björn Windshügel / Alexey G Kikhney / Ingrid B Müller / Raphael J Eberle / Dominik Oberthür / Huaixing Cang / Dmitri I Svergun / Markus Perbandt / ...Authors: Julia Drebes / Madeleine Künz / Björn Windshügel / Alexey G Kikhney / Ingrid B Müller / Raphael J Eberle / Dominik Oberthür / Huaixing Cang / Dmitri I Svergun / Markus Perbandt / Christian Betzel / Carsten Wrenger /
Abstract: Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a ...Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.
History
DepositionJul 9, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 23, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 7, 2018Group: Advisory / Data collection / Category: diffrn_source / pdbx_unobs_or_zero_occ_atoms / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2May 8, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / struct_conn / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_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_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hydroxyethylthiazole kinase
B: Hydroxyethylthiazole kinase
C: Hydroxyethylthiazole kinase
E: Hydroxyethylthiazole kinase
D: Hydroxyethylthiazole kinase
F: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,84517
Polymers178,9676
Non-polymers87811
Water9,116506
1
A: Hydroxyethylthiazole kinase
B: Hydroxyethylthiazole kinase
C: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,95910
Polymers89,4833
Non-polymers4767
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6590 Å2
ΔGint-59 kcal/mol
Surface area27440 Å2
MethodPISA
2
E: Hydroxyethylthiazole kinase
D: Hydroxyethylthiazole kinase
F: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,8867
Polymers89,4833
Non-polymers4034
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6280 Å2
ΔGint-37 kcal/mol
Surface area27700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.345, 62.483, 109.167
Angle α, β, γ (deg.)92.64, 92.05, 101.46
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23E
14A
24D
15A
25F
16B
26C
17B
27E
18B
28D
19B
29F
110C
210E
111C
211D
112C
212F
113E
213D
114E
214F
115D
215F

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: MET / Beg label comp-ID: MET / Refine code: _

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11VALVALAA1 - 2621 - 262
21VALVALBB1 - 2621 - 262
12VALVALAA1 - 2621 - 262
22VALVALCC1 - 2621 - 262
13GLUGLUAA1 - 2641 - 264
23GLUGLUED1 - 2641 - 264
14ILEILEAA1 - 2591 - 259
24ILEILEDE1 - 2591 - 259
15GLUGLUAA1 - 2611 - 261
25GLUGLUFF1 - 2611 - 261
16VALVALBB1 - 2621 - 262
26VALVALCC1 - 2621 - 262
17GLUGLUBB1 - 2611 - 261
27GLUGLUED1 - 2611 - 261
18ILEILEBB1 - 2591 - 259
28ILEILEDE1 - 2591 - 259
19GLUGLUBB1 - 2611 - 261
29GLUGLUFF1 - 2611 - 261
110VALVALCC1 - 2621 - 262
210VALVALED1 - 2621 - 262
111ARGARGCC1 - 2581 - 258
211ARGARGDE1 - 2581 - 258
112GLUGLUCC1 - 2611 - 261
212GLUGLUFF1 - 2611 - 261
113ILEILEED1 - 2591 - 259
213ILEILEDE1 - 2591 - 259
114GLNGLNED1 - 2601 - 260
214GLNGLNFF1 - 2601 - 260
115ILEILEDE1 - 2591 - 259
215ILEILEFF1 - 2591 - 259

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

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Components

#1: Protein
Hydroxyethylthiazole kinase / 4-methyl-5-beta-hydroxyethylthiazole kinase / Thz kinase


Mass: 29827.801 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus subsp. aureus MRSA252 (bacteria)
Gene: thiM, SAR2181 / Plasmid: pASK-IBA3 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6GEY3, hydroxyethylthiazole kinase
#2: Chemical
ChemComp-51F / 2-(2-methyl-1H-imidazol-1-yl)ethanol


Mass: 126.156 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C6H10N2O
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 506 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.32 Å3/Da / Density % sol: 47.07 %
Crystal growTemperature: 293.2 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 18 - 22 % PEG 3,350 (w/v), 0.2 M magnesium formate, 5 % isopropanol (v/v), soaked with 20 mM substrate analog 2-(2-methyl-1H-imidazole-1-yl)ethanol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jun 25, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.62→30 Å / Num. obs: 191461 / % possible obs: 93.4 % / Redundancy: 3.6 % / Net I/σ(I): 10.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0049refinement
XDSdata reduction
SCALAdata scaling
MOLREPphasing
RefinementResolution: 1.62→30 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.957 / SU B: 3.973 / SU ML: 0.069 / Cross valid method: THROUGHOUT / ESU R: 0.091 / ESU R Free: 0.087 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2028 9508 5 %RANDOM
Rwork0.18238 ---
obs0.1834 181567 93.19 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 29.176 Å2
Baniso -1Baniso -2Baniso -3
1-0.81 Å20.11 Å2-1.1 Å2
2---1.12 Å2-0.87 Å2
3---0.57 Å2
Refinement stepCycle: 1 / Resolution: 1.62→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11453 0 59 506 12018
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.01911492
X-RAY DIFFRACTIONr_bond_other_d0.0090.0211290
X-RAY DIFFRACTIONr_angle_refined_deg1.6991.9815644
X-RAY DIFFRACTIONr_angle_other_deg1.452325845
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.89351523
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.18726.148431
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.819151854
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.2851532
X-RAY DIFFRACTIONr_chiral_restr0.1110.21940
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.0213153
X-RAY DIFFRACTIONr_gen_planes_other0.0090.022362
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.5851.1176125
X-RAY DIFFRACTIONr_mcbond_other1.5811.1166124
X-RAY DIFFRACTIONr_mcangle_it2.4541.667637
X-RAY DIFFRACTIONr_mcangle_other2.4541.6617638
X-RAY DIFFRACTIONr_scbond_it2.3721.3455367
X-RAY DIFFRACTIONr_scbond_other2.3721.3455368
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.5491.9148008
X-RAY DIFFRACTIONr_long_range_B_refined5.8789.42113203
X-RAY DIFFRACTIONr_long_range_B_other5.8679.18512989
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A152430.08
12B152430.08
21A151260.08
22C151260.08
31A152690.08
32E152690.08
41A148250.07
42D148250.07
51A151120.09
52F151120.09
61B153090.07
62C153090.07
71B150810.09
72E150810.09
81B146780.07
82D146780.07
91B152600.09
92F152600.09
101C150780.08
102E150780.08
111C145580.08
112D145580.08
121C152810.07
122F152810.07
131E146490.08
132D146490.08
141E151080.08
142F151080.08
151D147380.08
152F147380.08
LS refinement shellResolution: 1.62→1.662 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.253 653 -
Rwork0.246 12627 -
obs--87.67 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.06260.00920.03090.2425-0.03920.27680.0399-0.0052-0.044-0.0782-0.03120.0342-0.00930.0407-0.00870.1263-0.00060.00010.1428-0.02290.2205-14.1838-26.817911.7181
20.17880.0593-0.03120.4936-0.13480.0903-0.027-0.01610.0369-0.00440.01120.015-0.0111-0.01230.01580.0734-0.00640.00220.1483-0.00530.2473-17.71420.94730.2355
30.45350.0393-0.07010.1592-0.06080.0874-0.01130.01650.00680.02590.0334-0.05350.00570.0039-0.02210.0613-0.0235-0.00020.1726-0.01970.248.9633-17.689733.259
40.40830.48770.67090.67310.88451.1875-0.13230.0050.0565-0.0939-0.05280.1305-0.1748-0.03990.18510.1479-0.0092-0.0510.1478-0.05910.19011.86466.8638-41.4855
50.67230.33430.75840.72711.13961.90050.21420.1390.16170.4631-0.31410.01430.7035-0.35380.09980.4049-0.20240.00510.29670.03180.17967.8494.569-9.5875
60.2791-0.00610.23621.59380.57580.4421-0.23470.02310.0515-0.20270.02150.1901-0.34360.02170.21310.3545-0.0302-0.2140.027-0.00870.264412.641233.5871-24.0357
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 264
2X-RAY DIFFRACTION2B1 - 262
3X-RAY DIFFRACTION3C1 - 262
4X-RAY DIFFRACTION4E1 - 269
5X-RAY DIFFRACTION5D1 - 259
6X-RAY DIFFRACTION6F1 - 261

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