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- PDB-5cga: Structure of Hydroxyethylthiazole kinase ThiM from Staphylococcus... -

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Basic information

Entry
Database: PDB / ID: 5cga
TitleStructure of Hydroxyethylthiazole kinase ThiM from Staphylococcus aureus in complex with substrate analog 2-(1,3,5-trimethyl-1H-pyrazole-4-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 / phosphorylation / 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-(1,3,5-trimethyl-1H-pyrazol-4-yl)ethanol / Hydroxyethylthiazole kinase
Similarity search - Component
Biological speciesStaphylococcus aureus MRSA252 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.87 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_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_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
F: Hydroxyethylthiazole kinase
H: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,87516
Polymers178,8536
Non-polymers1,02210
Water8,755486
1
A: Hydroxyethylthiazole kinase
B: Hydroxyethylthiazole kinase
C: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,9629
Polymers89,4263
Non-polymers5366
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6090 Å2
ΔGint-54 kcal/mol
Surface area26830 Å2
MethodPISA
2
E: Hydroxyethylthiazole kinase
F: Hydroxyethylthiazole kinase
H: Hydroxyethylthiazole kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,9137
Polymers89,4263
Non-polymers4874
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5940 Å2
ΔGint-37 kcal/mol
Surface area26970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.026, 62.362, 108.320
Angle α, β, γ (deg.)92.59, 91.39, 101.28
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23E
14A
24F
15A
25H
16B
26C
17B
27E
18B
28F
19B
29H
110C
210E
111C
211F
112C
212H
113E
213F
114E
214H
115F
215H

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11METMETGLUGLUAA1 - 2611 - 261
21METMETGLUGLUBB1 - 2611 - 261
12METMETGLUGLUAA1 - 2611 - 261
22METMETGLUGLUCC1 - 2611 - 261
13METMETGLUGLUAA1 - 2641 - 264
23METMETGLUGLUED1 - 2641 - 264
14ASNASNARGARGAA2 - 2582 - 258
24ASNASNARGARGFE2 - 2582 - 258
15METMETARGARGAA1 - 2581 - 258
25METMETARGARGHF1 - 2581 - 258
16METMETVALVALBB1 - 2621 - 262
26METMETVALVALCC1 - 2621 - 262
17METMETGLUGLUBB1 - 2611 - 261
27METMETGLUGLUED1 - 2611 - 261
18ASNASNARGARGBB2 - 2582 - 258
28ASNASNARGARGFE2 - 2582 - 258
19METMETARGARGBB1 - 2581 - 258
29METMETARGARGHF1 - 2581 - 258
110METMETGLUGLUCC1 - 2611 - 261
210METMETGLUGLUED1 - 2611 - 261
111ASNASNARGARGCC2 - 2582 - 258
211ASNASNARGARGFE2 - 2582 - 258
112METMETARGARGCC1 - 2581 - 258
212METMETARGARGHF1 - 2581 - 258
113ASNASNARGARGED2 - 2582 - 258
213ASNASNARGARGFE2 - 2582 - 258
114METMETARGARGED1 - 2581 - 258
214METMETARGARGHF1 - 2581 - 258
115ASNASNARGARGFE2 - 2582 - 258
215ASNASNARGARGHF2 - 2582 - 258

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: 29808.758 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus MRSA252 (bacteria)
Strain: MRSA252 / Gene: thiM, SAR2181 / Plasmid: pASK-IBA3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BLR / References: UniProt: Q6GEY3, hydroxyethylthiazole kinase
#2: Chemical
ChemComp-KP6 / 2-(1,3,5-trimethyl-1H-pyrazol-4-yl)ethanol


Mass: 154.210 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H14N2O
#3: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 18.015 Da / Num. of mol.: 486 / 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: 2.29 Å3/Da / Density % sol: 46.34 %
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 25 mM substrate analog 2-(1,3,5-trimethyl-1H-pyrazole-4-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: 0.976262 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 15, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976262 Å / Relative weight: 1
ReflectionResolution: 1.87→30 Å / Num. obs: 125936 / % possible obs: 95.8 % / Redundancy: 3.6 % / Net I/σ(I): 12.7

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Processing

Software
NameVersionClassification
REFMAC5.7.0032refinement
XDSdata reduction
SCALAdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.87→29.81 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.95 / SU B: 5.377 / SU ML: 0.08 / Cross valid method: THROUGHOUT / ESU R: 0.128 / ESU R Free: 0.119 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.19899 6337 5 %RANDOM
Rwork0.16862 ---
obs0.1701 119598 95.79 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 35.128 Å2
Baniso -1Baniso -2Baniso -3
1--0.53 Å2-0.49 Å2-0.37 Å2
2---0.89 Å2-0.27 Å2
3---1.6 Å2
Refinement stepCycle: 1 / Resolution: 1.87→29.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11228 0 70 486 11784
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.01911124
X-RAY DIFFRACTIONr_bond_other_d0.010.0210898
X-RAY DIFFRACTIONr_angle_refined_deg1.7381.98115169
X-RAY DIFFRACTIONr_angle_other_deg1.453324895
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.75751485
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.00226.064404
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.63151735
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.5031529
X-RAY DIFFRACTIONr_chiral_restr0.1160.21887
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.02112771
X-RAY DIFFRACTIONr_gen_planes_other0.0090.022273
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_it1.6821.4035978
X-RAY DIFFRACTIONr_mcbond_other1.681.4035977
X-RAY DIFFRACTIONr_mcangle_it2.4742.0877451
X-RAY DIFFRACTIONr_mcangle_other2.4742.0887452
X-RAY DIFFRACTIONr_scbond_it2.3251.6465146
X-RAY DIFFRACTIONr_scbond_other2.3251.6465147
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.4112.3737719
X-RAY DIFFRACTIONr_long_range_B_refined5.84712.18412886
X-RAY DIFFRACTIONr_long_range_B_other5.78811.88312683
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)
11A149840.06
12B149840.06
21A150440.06
22C150440.06
31A150420.06
32E150420.06
41A143910.05
42F143910.05
51A149910.05
52H149910.05
61B154790.05
62C154790.05
71B147940.07
72E147940.07
81B145020.05
82F145020.05
91B151780.06
92H151780.06
101C148890.07
102E148890.07
111C144720.05
112F144720.05
121C152520.04
122H152520.04
131E142610.06
132F142610.06
141E148260.06
142H148260.06
151F145090.05
152H145090.05
LS refinement shellResolution: 1.87→1.918 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.307 526 -
Rwork0.287 8713 -
obs--94.99 %
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)
10.98350.0818-0.05931.324-0.24651.59470.01180.1844-0.1016-0.30930.01490.13520.0235-0.0368-0.02680.16140.0401-0.04830.0866-0.05670.0569-6.231-11.862-12.986
21.08170.1881-0.15862.0755-0.59211.0998-0.0125-0.07520.20740.0468-0.00680.0535-0.1398-0.00240.01920.0550.0421-0.03240.0521-0.04610.0678-10.12415.0955.292
31.93050.321-0.1451.1173-0.13450.9297-0.01710.0357-0.1335-0.05360.0343-0.24020.09010.159-0.01720.04290.048-0.01380.1066-0.05740.078417.191-3.0738.39
41.55940.77441.43112.4041.48752.2553-0.13820.00130.106-0.0643-0.14830.3441-0.254-0.13240.28640.27670.0379-0.03450.2432-0.10480.11699.43121.903-65.643
52.62940.4184-0.05012.14720.63182.32350.1083-0.41220.00170.8111-0.25880.15910.2893-0.31120.15050.5937-0.070.08140.2289-0.08980.154115.4419.696-33.669
61.5336-0.44040.1393.41930.9771.5893-0.17310.15580.2279-0.2416-0.11060.1541-0.5335-0.05910.28360.52980.0169-0.16280.1365-0.06630.182320.11448.598-48.826
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 262
2X-RAY DIFFRACTION2B1 - 262
3X-RAY DIFFRACTION3C1 - 262
4X-RAY DIFFRACTION4E1 - 266
5X-RAY DIFFRACTION5F1 - 260
6X-RAY DIFFRACTION6H1 - 259

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