Kanamycin nucleotidyltransferase

 

Kanamycin nucleotidyltransferase (KNTase) is a plasmid encoded enzyme responsible for some types of bacterial resistance to aminoglycosides. It catalyses the transfer of a nucleoside monophosphate group onto the 4' hydroxyl group of kanamycin, causing deactivation of this drug. The enzyme is also capable of deactivating many other drugs including neomycins, and can utilise ATP, GTP, CTP, TTP or UTP as the NMP donor.

 

Reference Protein and Structure

Sequence
P05057 UniProt (2.7.7.-) IPR012481 (Sequence Homologues) (PDB Homologues)
Biological species
Staphylococcus aureus (Bacteria) Uniprot
PDB
1kny - KANAMYCIN NUCLEOTIDYLTRANSFERASE (2.5 Å) PDBe PDBsum 1kny
Catalytic CATH Domains
3.30.460.10 CATHdb 1.20.120.330 CATHdb (see all for 1kny)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.-)

ATP(4-)
CHEBI:30616ChEBI
+
kanamycin A
CHEBI:17630ChEBI
→
diphosphate(3-)
CHEBI:33019ChEBI
+
4'-adenylkanamycin A
CHEBI:137400ChEBI

Enzyme Mechanism

Introduction

Kanamycin nucleotidyltransferase catalyses a single in-line displacement attack by the 4' hydroxyl group of kanamycin on the alpha phosphate of the NTP. The leaving group is pyrophosphate. As is typical for enzymatic phosphodiester reactions, the substitution is concerted with a slightly associative transition-state. Glu 145 acts as a base to deprotonate the attacking 4' hydroxyl group, while Lys 149 functions to stabilise the accumulation of negative charge on the alpha phosphate oxygens in the transition state. An Mg2+ ion coordinates the beta and gamma phosphates, so stabilising the departing pyrophosphate leaving group. Deprotonation of Glu 145 can occur to regenerate the active site ready for another round of catalysis such as a water molecule picking the proton up.

Catalytic Residues Roles

UniProt PDB* (1kny)
Glu52, Asp50 (main-C) Glu52B, Asp50B (main-C) Proposed to coordinate to a magnesium ion which in turn stabilises the pyrophosphate leaving group. metal ligand
Glu145 Glu145A Acts as a general base to deprotonate the 4' hydroxyl group of the antibiotic as it attacks the alpha phosphate of the NTP. activator, proton acceptor, proton donor
Lys149 Lys149A Provides a positive charge to stabilise accumulation of negative charge on the alpha phosphate oxygens in the transition state. electrostatic stabiliser, polar interaction
*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

proton transfer, bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Gerratana B et al. (2001), Biochemistry, 40, 2972-2977. Characterization of the Transition-State Structure of the Reaction of Kanamycin Nucleotidyltransferase by Heavy-Atom Kinetic Isotope Effects†. DOI:10.1021/bi002557x. PMID:11258909.
  2. Cleland WW et al. (2006), Chem Rev, 106, 3252-3278. Enzymatic Mechanisms of Phosphate and Sulfate Transfer. DOI:10.1021/cr050287o. PMID:16895327.
  3. Chen-Goodspeed M et al. (1999), Bioorg Chem, 27, 395-408. Kinetic Mechanism of Kanamycin Nucleotidyltransferase from Staphylococcus aureus. DOI:10.1006/bioo.1999.1144.
  4. Pedersen LC et al. (1995), Biochemistry, 34, 13305-13311. Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase. DOI:10.1021/bi00041a005. PMID:7577914.

Step 1. Glu45 abstracts the proton on the 4' hydroxyl group in kanamycin. This activates the group for nucleophilic attack of the alpha phosphate on ATP. Lys149 stabilises the negatively charged transition state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys149A electrostatic stabiliser
Glu52B metal ligand
Asp50B (main-C) metal ligand
Glu145A activator
Lys149A polar interaction
Glu145A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed

Step 2. Solvent water molecule deprotonates Glu145 to regenerate active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu145A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu45 abstracts the proton on the 4' hydroxyl group in kanamycin. This activates the group for nucleophilic attack of the alpha phosphate on ATP. Lys149 stabilises the negatively charged transition state.

Step 2. Solvent water molecule deprotonates Glu145 to regenerate active site.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Morwenna Hall