Kanamycin kinase

 

Aminoglycosides are potent antibiotics which bind to the prokaryotic 16S ribosomal subunit, inhibiting protein synthesis. Resistance to these antibiotics however has developed in many bacterial species due to the enzyme aminoglycoside kinase which is able to catalyse the phosphorylation of the aminoglycoside resulting in its inactivation. Kinetic and structural studies of this enzyme therefore are vital in prevention and understanding of antibiotic resistance. The enzyme shows structural alignment, despite very low sequence conservation, to eukaryotic protein kinases indicating a possible evolutionary relationship.

 

Reference Protein and Structure

Sequence
P0A3Y5 UniProt (2.7.1.95) IPR024165 (Sequence Homologues) (PDB Homologues)
Biological species
Enterococcus faecalis (Bacteria) Uniprot
PDB
1l8t - Crystal Structure Of 3',5""-Aminoglycoside Phosphotransferase Type IIIa ADP Kanamycin A Complex (2.4 Å) PDBe PDBsum 1l8t
Catalytic CATH Domains
3.90.1200.10 CATHdb 3.30.200.20 CATHdb (see all for 1l8t)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:2.7.1.95)

kanamycin A(4+)
CHEBI:58214ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
ADP(3-)
CHEBI:456216ChEBI
+
kanamycin A 3'-phosphate(2+)
CHEBI:57909ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Kanamycin kinase (phosphorylating), Neomycin phosphotransferase, Neomycin-kanamycin phosphotransferase, Aminoglycoside 3'-phosphotransferase, APH(3'),

Enzyme Mechanism

Introduction

Nucleophilic attack on the gamma phosphate of ATP by the 3' or 5' OH group of the substrate allows formation of a pentavalent phosphate transition state which collapses to release the products. In order to accelerate this the deprotonation of the OH group is accomplished by Asp 190 and the transition state is stabilised by contact with Lys 44.

Catalytic Residues Roles

UniProt PDB* (1l8t)
Asp208, Asn195 Asp208(207)A, Asn195(194)A Coordinate to magnesium ions which in turn coordinate and stabilise the phosphate groups on ATP for phosphoryl transfer. metal ligand
Lys44 Lys44(43)A Contacts the beta and gamma phosphates of ATP, thus is positioned to stabilise the negative charge that builds up when the pentavalent phosphate transition state forms. electrostatic stabiliser, polar interaction
Asp190 Asp190(189)A Acts to deprotonate the attacking OH group to allow it to act as a nucleophile and form a bond to the gamma phosphate of ATP. proton shuttle (general acid/base), proton acceptor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, proton transfer

References

  1. Hon WC et al. (1997), Cell, 89, 887-895. Structure of an Enzyme Required for Aminoglycoside Antibiotic Resistance Reveals Homology to Eukaryotic Protein Kinases. DOI:10.1016/s0092-8674(00)80274-3. PMID:9200607.
  2. Power BH et al. (2017), Chem Biol Drug Des, 89, 84-97. Insight into the mechanism of chemical modification of antibacterial agents by antibiotic resistance enzyme O-phosphotransferase-IIIA. DOI:10.1111/cbdd.12835. PMID:27495974.
  3. Thompson PR et al. (2002), Biochemistry, 41, 7001-7007. Mechanism of Aminoglycoside Antibiotic Kinase APH(3‘)-IIIa:  Role of the Nucleotide Positioning Loop†. DOI:10.1021/bi0256680.
  4. Wright GD (1999), Front Biosci, 4, d9-. Aminoglycoside phosphotransferases: proteins, structure, and mechanism. DOI:10.2741/Wright.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys44(43)A electrostatic stabiliser
Asp190(189)A proton shuttle (general acid/base)
Asn195(194)A metal ligand
Asp208(207)A metal ligand
Lys44(43)A polar interaction
Asp190(189)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer

Step 1.

Contributors

Peter Sarkies, Gemma L. Holliday, Morwenna Hall