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- PDB-6hwl: Glucosamine kinase in complex with glucosamine, ADP and inorganic... -

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Basic information

Entry
Database: PDB / ID: 6hwl
TitleGlucosamine kinase in complex with glucosamine, ADP and inorganic phosphate
ComponentsGlucosamine kinase
KeywordsTRANSFERASE / Glucosamine kinase
Function / homology
Function and homology information


glucosamine kinase / glucosamine kinase activity / carbohydrate metabolic process / phosphorylation / magnesium ion binding / ATP binding
Similarity search - Function
Glucosamine kinase / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / 2-amino-2-deoxy-beta-D-glucopyranose / DI(HYDROXYETHYL)ETHER / PHOSPHATE ION / Glucosamine kinase
Similarity search - Component
Biological speciesStreptacidiphilus jiangxiensis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.148 Å
AuthorsManso, J.A. / Pereira, P.J.B.
CitationJournal: mBio / Year: 2019
Title: Molecular Fingerprints for a Novel Enzyme Family in with Glucosamine Kinase Activity.
Authors: José A Manso / Daniela Nunes-Costa / Sandra Macedo-Ribeiro / Nuno Empadinhas / Pedro José Barbosa Pereira /
Abstract: have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has ... have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in and can be explored to enhance antibiotic production. The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in , nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.
History
DepositionOct 12, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 8, 2020Group: Data collection / Category: chem_comp / Item: _chem_comp.type
Revision 1.3Jul 29, 2020Group: Data collection / Derived calculations ...Data collection / Derived calculations / Refinement description / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / software / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _software.name / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.4Sep 30, 2020Group: Database references / Structure summary / Category: chem_comp / citation / citation_author
Item: _chem_comp.pdbx_synonyms / _citation.title / _citation_author.identifier_ORCID
Revision 1.5May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glucosamine kinase
B: Glucosamine kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,96613
Polymers96,6172
Non-polymers1,34811
Water4,378243
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The glucosamine kinase is a monomer as determined by SAXS and gel-filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3410 Å2
ΔGint-47 kcal/mol
Surface area35530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.764, 97.435, 80.188
Angle α, β, γ (deg.)90.00, 107.42, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein / Sugars , 2 types, 3 molecules AB

#1: Protein Glucosamine kinase / Maltokinase


Mass: 48308.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptacidiphilus jiangxiensis (bacteria)
Gene: SAMN05414137_114149 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1H7TQR5
#8: Sugar ChemComp-GCS / 2-amino-2-deoxy-beta-D-glucopyranose / beta-D-glucosamine / 2-amino-2-deoxy-beta-D-glucose / 2-amino-2-deoxy-D-glucose / 2-amino-2-deoxy-glucose / D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 179.171 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H13NO5
IdentifierTypeProgram
DGlcpNbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 7 types, 253 molecules

#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-BTB / 2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / BIS-TRIS BUFFER


Mass: 209.240 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H19NO5 / Comment: pH buffer*YM
#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 243 / 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: 2.27 Å3/Da / Density % sol: 45.84 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 100 mM Bis-Tris pH 6.1, 16% (wt/vol) PEG 3350, 0.15 M MgCl2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-3 / Wavelength: 0.9677 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Mar 2, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 2.148→60.175 Å / Num. obs: 46229 / % possible obs: 98.3 % / Redundancy: 7 % / Biso Wilson estimate: 40.8 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.045 / Rrim(I) all: 0.122 / Net I/σ(I): 11.8
Reflection shellResolution: 2.15→2.19 Å / Redundancy: 7.3 % / Rmerge(I) obs: 1.543 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 2340 / CC1/2: 0.559 / Rpim(I) all: 0.608 / Rrim(I) all: 1.66 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
XDSdata reduction
autoPROCdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: D_1200012387

Resolution: 2.148→39.888 Å / SU ML: 0.27 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 25.71
RfactorNum. reflection% reflection
Rfree0.243 2320 5.02 %
Rwork0.2071 --
obs0.2089 46219 98.28 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.148→39.888 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5984 0 83 243 6310
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0026209
X-RAY DIFFRACTIONf_angle_d0.4988521
X-RAY DIFFRACTIONf_dihedral_angle_d12.8993670
X-RAY DIFFRACTIONf_chiral_restr0.039998
X-RAY DIFFRACTIONf_plane_restr0.0041110
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1484-2.19230.31231370.28812601X-RAY DIFFRACTION99
2.1923-2.23990.33361340.27292593X-RAY DIFFRACTION99
2.2399-2.2920.3061430.26482618X-RAY DIFFRACTION99
2.292-2.34940.26771360.24812564X-RAY DIFFRACTION99
2.3494-2.41290.29111350.24592589X-RAY DIFFRACTION99
2.4129-2.48390.2731260.23572598X-RAY DIFFRACTION99
2.4839-2.5640.26111460.23192598X-RAY DIFFRACTION99
2.564-2.65560.28251520.23432571X-RAY DIFFRACTION99
2.6556-2.76190.30091300.23312594X-RAY DIFFRACTION99
2.7619-2.88760.26611170.22982573X-RAY DIFFRACTION98
2.8876-3.03980.261510.22182317X-RAY DIFFRACTION89
3.0398-3.23020.25581200.21742463X-RAY DIFFRACTION93
3.2302-3.47940.2511330.19972638X-RAY DIFFRACTION100
3.4794-3.82930.21481360.19032626X-RAY DIFFRACTION100
3.8293-4.38290.1991420.17432635X-RAY DIFFRACTION100
4.3829-5.51960.2181490.17882629X-RAY DIFFRACTION100
5.5196-39.89450.2321330.2022692X-RAY DIFFRACTION100
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)
11.75170.9643-0.13345.02950.7691.7920.0753-0.07690.19620.2811-0.05110.0315-0.2436-0.1014-0.02250.2386-0.00040.00780.248-0.01590.260462.781846.275684.6915
21.733-0.38030.07931.05730.11221.46740.0308-0.1453-0.09890.1675-0.08080.01880.0223-0.20930.04840.3138-0.0237-0.0050.2739-0.00090.330441.835421.856284.7011
31.14391.5138-0.60743.1042-1.14032.75190.0181-0.376-0.14250.28440.08070.06640.4334-0.9379-0.12370.433-0.10660.03310.8280.09990.464229.8588-24.0199110.9319
40.88050.0289-0.1221.7903-0.13153.6630.0103-0.2320.06820.252-0.0045-0.0968-0.39240.0663-0.01280.3561-0.0084-0.03740.31860.03460.336148.4233-9.8191100.4747
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 193 )
2X-RAY DIFFRACTION2chain 'A' and (resid 194 through 433 )
3X-RAY DIFFRACTION3chain 'B' and (resid 4 through 211 )
4X-RAY DIFFRACTION4chain 'B' and (resid 212 through 441 )

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