PDBsum entry 1l8t

Go to PDB code: 
protein ligands metals links
Transferase PDB id
Protein chain
263 a.a. *
_MG ×2
Waters ×65
* Residue conservation analysis
PDB id:
Name: Transferase
Title: Crystal structure of 3',5"-aminoglycoside phosphotransferase type iiia adp kanamycin a complex
Structure: Aminoglycoside 3'-phosphotransferase. Chain: a. Synonym: kanamycin kinase type iii, neomycin-kanamycin phosphotransferase type iii, aph(3')iii. Engineered: yes
Source: Enterococcus faecalis. Organism_taxid: 1351. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Monomer (author assigned)
2.40Å     R-factor:   0.234     R-free:   0.291
Authors: D.H.Fong,A.M.Berghuis
Key ref:
D.H.Fong and A.M.Berghuis (2002). Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry. EMBO J, 21, 2323-2331. PubMed id: 12006485 DOI: 10.1093/emboj/21.10.2323
21-Mar-02     Release date:   19-Jun-02    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P0A3Y5  (KKA3_ENTFL) -  Aminoglycoside 3'-phosphotransferase
264 a.a.
263 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Kanamycin kinase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP + kanamycin = ADP + kanamycin 3'-phosphate
+ kanamycin
Bound ligand (Het Group name = ADP)
corresponds exactly
kanamycin 3'-phosphate
Bound ligand (Het Group name = KAN)
matches with 89.00% similarity
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     response to antibiotic   2 terms 
  Biochemical function     nucleotide binding     7 terms  


DOI no: 10.1093/emboj/21.10.2323 EMBO J 21:2323-2331 (2002)
PubMed id: 12006485  
Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry.
D.H.Fong, A.M.Berghuis.
The misuse of antibiotics has selected for bacteria that have evolved mechanisms for evading the effects of these drugs. For aminoglycosides, a group of clinically important bactericidal antibiotics that target the A-site of the 16S ribosomal RNA, the most common mode of resistance is enzyme-catalyzed chemical modification of the drug. While aminoglycosides are structurally diverse, a single enzyme can confer resistance to many of these antibiotics. For example, the aminoglycoside kinase APH(3')-IIIa, produced by pathogenic Gram-positive bacteria such as enterococci and staphylococci, is capable of detoxifying at least 10 distinct aminoglycosides. Here we describe the crystal structures of APH(3')-IIIa in complex with ADP and kanamycin A or neomycin B. These structures reveal that the basis for this enzyme's substrate promiscuity is the presence of two alternative subsites in the antibiotic binding pocket. Furthermore, comparison between the A-site of the bacterial ribosome and APH(3')-IIIa shows that mimicry is the second major factor in dictating the substrate spectrum of APH(3')-IIIa. These results suggest a potential strategy for drug design aimed at circumventing antibiotic resistance.
  Selected figure(s)  
Figure 2.
Figure 2 (A) Simulated annealing F[o]-F[c] omit map for kanamycin A (contoured at 2 ). Residues forming hydrogen bond interactions are shown. (B) Simulated annealing F[o]-F[c] omit map for neomycin B (contoured at 2 ), also showing residues that form hydrogen bond interactions with this antibiotic.
Figure 4.
Figure 4 Comparison of aminoglycoside binding to APH(3')-IIIa versus the bacterial ribosome. (A) Superposition of neomycin B, in the conformation observed in the APH(3')-IIIa ternary complex, and paromomycin I in the conformation observed in the crystal structure of the 30S ribosomal subunit (Carter et al., 2000). Neomycin B is shown in solid colors and paromomycin I is semi-transparent. (B) Schematic overview of hydrogen bond interactions made by aminoglycosides with APH(3')-IIIa and the bacterial ribosome (Fourmy et al., 1998; Yoshizawa et al., 1998; Carter et al., 2000). A combined generic chemical structure for 4,6- and 4,5-disubstituted aminoglycosides is shown, highlighting common functional groups (see also Table I). Hydrogen bond interactions made by APH(3')-IIIa with aminoglycosides are shown in white boxes, while those made by the ribosome, as observed in the ribosome‚ąíparomomycin I crystal structure (Carter et al., 2000), are shown in aqua. Additional interactions made by the ribosome, as observed in NMR studies of neomycin B and gentamycin C[1a] (Fourmy et al., 1998; Yoshizawa et al., 1998), are displayed in yellow boxes. (C) Stereo view of the van der Waals surface of the APH(3')-IIIa aminoglycoside-binding pocket. Also shown are kanamycin A and neomycin B. (D) Stereo view of the van der Waals surface of the bacterial ribosomal aminoglycoside-binding pocket. Also shown are paromomycin I and a modeled kanamycin A. This stereo view has been subjected to a 180° rotation around the vertical axis with respect to (C), to show that opposite faces of the aminoglycosides form predominant van der Waals interactions with either the ribosome or APH(3')-IIIa.
  The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 2323-2331) copyright 2002.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20089863 D.H.Fong, C.T.Lemke, J.Hwang, B.Xiong, and A.M.Berghuis (2010).
Structure of the antibiotic resistance factor spectinomycin phosphotransferase from Legionella pneumophila.
  J Biol Chem, 285, 9545-9555.
PDB codes: 3i0o 3i0q 3i1a
20077512 E.D.Scheeff, H.L.Axelrod, M.D.Miller, H.J.Chiu, A.M.Deacon, I.A.Wilson, and G.Manning (2010).
Genomics, evolution, and crystal structure of a new family of bacterial spore kinases.
  Proteins, 78, 1470-1482.
PDB code: 2q83
19898995 E.H.Serpersu, C.Ozen, A.L.Norris, C.Steren, and N.Whittemore (2010).
Backbone resonance assignments of a promiscuous aminoglycoside antibiotic resistance enzyme; the aminoglycoside phosphotransferase(3')-IIIa.
  Biomol NMR Assign, 4, 9.  
20822442 M.Morar, and G.D.Wright (2010).
The genomic enzymology of antibiotic resistance.
  Annu Rev Genet, 44, 25-51.  
20833577 M.S.Ramirez, and M.E.Tolmasky (2010).
Aminoglycoside modifying enzymes.
  Drug Resist Updat, 13, 151-171.  
20556826 M.Toth, H.Frase, N.T.Antunes, C.A.Smith, and S.B.Vakulenko (2010).
Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2''-IVa.
  Protein Sci, 19, 1565-1576.
PDB codes: 3n4t 3n4u 3n4v
  20057078 M.Toth, S.Vakulenko, and C.A.Smith (2010).
Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2''-phosphotransferase-IVa.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 66, 81-84.  
20221476 S.Hanessian, and J.P.Maianti (2010).
Biomimetic synthesis and structural refinement of the macrocyclic dimer aminoglycoside 66-40C--the remarkably selective self-condensation of a putative aldehyde intermediate in the submerged culture medium producing sisomicin.
  Chem Commun (Camb), 46, 2013-2015.  
19548090 D.Bandyopadhyay, J.Huan, J.Prins, J.Snoeyink, W.Wang, and A.Tropsha (2009).
Identification of family-specific residue packing motifs and their use for structure-based protein function prediction: II. Case studies and applications.
  J Comput Aided Mol Des, 23, 785-797.  
19433564 D.H.Fong, and A.M.Berghuis (2009).
Structural basis of APH(3')-IIIa-mediated resistance to N1-substituted aminoglycoside antibiotics.
  Antimicrob Agents Chemother, 53, 3049-3055.
PDB codes: 3h8p 3tm0
19629142 J.Zhang, K.Keller, J.Y.Takemoto, M.Bensaci, A.Litke, P.G.Czyryca, and C.W.Chang (2009).
Synthesis and combinational antibacterial study of 5''-modified neomycin.
  J Antibiot (Tokyo), 62, 539-544.  
19437437 L.Wu, and E.H.Serpersu (2009).
Deciphering interactions of the aminoglycoside phosphotransferase(3')-IIIa with its ligands.
  Biopolymers, 91, 801-809.  
19429619 P.G.Young, R.Walanj, V.Lakshmi, L.J.Byrnes, P.Metcalf, E.N.Baker, S.B.Vakulenko, and C.A.Smith (2009).
The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] provide insights into substrate selectivity in the APH(2'') subfamily.
  J Bacteriol, 191, 4133-4143.
PDB codes: 3ham 3hav
19558703 R.Alterovitz, A.Arvey, S.Sankararaman, C.Dallett, Y.Freund, and K.Sjölander (2009).
ResBoost: characterizing and predicting catalytic residues in enzymes.
  BMC Bioinformatics, 10, 197.  
19012394 J.Zhang, F.I.Chiang, L.Wu, P.G.Czyryca, D.Li, and C.W.Chang (2008).
Surprising alteration of antibacterial activity of 5"-modified neomycin against resistant bacteria.
  J Med Chem, 51, 7563-7573.  
  18259066 L.J.Byrnes, A.Badarau, S.B.Vakulenko, and C.A.Smith (2008).
Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2''-phosphotransferase-Ic [APH(2'')-Ic] from Enterococcus gallinarum.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 64, 126-129.  
18174989 M.Hainrichson, I.Nudelman, and T.Baasov (2008).
Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases.
  Org Biomol Chem, 6, 227-239.  
18487349 S.Dutta, B.K.Kolli, A.Tang, S.Sassa, and K.P.Chang (2008).
Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin.
  Eukaryot Cell, 7, 1146-1157.  
  17671368 D.Iino, Y.Takakura, M.Kuroiwa, R.Kawakami, Y.Sasaki, T.Hoshino, K.Ohsawa, A.Nakamura, and S.Yajima (2007).
Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 63, 685-688.  
17870543 J.Li, F.I.Chiang, H.N.Chen, and C.W.Chang (2007).
Synthesis and antibacterial activity of pyranmycin derivatives with N-1 and O-6 modifications.
  Bioorg Med Chem, 15, 7711-7719.  
17561509 L.Hou, M.T.Honaker, L.M.Shireman, L.M.Balogh, A.G.Roberts, K.C.Ng, A.Nath, and W.M.Atkins (2007).
Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases.
  J Biol Chem, 282, 23264-23274.  
17418235 M.Kaul, C.M.Barbieri, A.R.Srinivasan, and D.S.Pilch (2007).
Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis.
  J Mol Biol, 369, 142-156.  
17563376 M.Korczynska, T.A.Mukhtar, G.D.Wright, and A.M.Berghuis (2007).
Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase.
  Proc Natl Acad Sci U S A, 104, 10388-10393.
PDB codes: 2z2n 2z2o 2z2p
17509130 O.Keskin (2007).
Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies.
  BMC Struct Biol, 7, 31.  
16481313 D.R.Tanner, J.D.Dewey, M.R.Miller, and A.R.Buskirk (2006).
Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1.
  J Biol Chem, 281, 10561-10566.  
  16511108 C.T.Lemke, J.Hwang, B.Xiong, N.P.Cianciotto, and A.M.Berghuis (2005).
Crystallization and preliminary crystallographic analysis of an aminoglycoside kinase from Legionella pneumophila.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 61, 606-608.  
15908922 C.Zhang, D.M.Kenski, J.L.Paulson, A.Bonshtien, G.Sessa, J.V.Cross, D.J.Templeton, and K.M.Shokat (2005).
A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.
  Nat Methods, 2, 435-441.  
  16511055 R.Walanj, P.Young, H.M.Baker, E.N.Baker, P.Metcalf, J.W.Chow, S.Lerner, S.Vakulenko, and C.A.Smith (2005).
Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2''-phosphotransferase-Ib [APH(2'')-Ib].
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 61, 410-413.  
15388945 D.H.Fong, and A.M.Berghuis (2004).
Crystallization and preliminary crystallographic analysis of 3'-aminoglycoside kinase type IIIa complexed with a eukaryotic protein kinase inhibitor, CKI-7.
  Acta Crystallogr D Biol Crystallogr, 60, 1897-1899.  
12925993 D.S.Pilch, M.Kaul, C.M.Barbieri, and J.E.Kerrigan (2003).
Thermodynamics of aminoglycoside-rRNA recognition.
  Biopolymers, 70, 58-79.  
12936992 K.J.Dery, B.Søballe, M.S.Witherspoon, D.Bui, R.Koch, D.J.Sherratt, and M.E.Tolmasky (2003).
The aminoglycoside 6'-N-acetyltransferase type Ib encoded by Tn1331 is evenly distributed within the cell's cytoplasm.
  Antimicrob Agents Chemother, 47, 2897-2902.  
12878003 L.C.James, and D.S.Tawfik (2003).
Conformational diversity and protein evolution--a 60-year-old hypothesis revisited.
  Trends Biochem Sci, 28, 361-368.  
12925992 Q.Vicens, and E.Westhof (2003).
Molecular recognition of aminoglycoside antibiotics by ribosomal RNA and resistance enzymes: an analysis of x-ray crystal structures.
  Biopolymers, 70, 42-57.  
12377564 T.Parkinson (2002).
The impact of genomics on anti-infectives drug discovery and development.
  Trends Microbiol, 10, S22-S26.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.