NMR-Guided Fragment-Based Approach for the Design of AAC(6′)-Ib Ligands (2025)

Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia revealed by crystallographic and small-angle X-ray scattering analysis

Acta crystallographica. Section D, Biological crystallography, 2014

Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal…

The use of aminoglycoside derivatives to study the mechanism of aminoglycoside 6′-N-acetyltransferase and the role of 6′-NH2 in antibacterial activity

Bioorganic & Medicinal Chemistry, 2007

Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6′-N-acetyltransferases (AAC(6′)s). We report here the regioselective and efficient synthesis of N-6′-acylated aminoglycosides and their use as probes to study AAC(6′)-Ii and aminoglycoside-RNA complexes. Our results emphasize the central role of N-6′ nucleophilicity for transformation by AAC(6′)-Ii and the importance of hydrogen bonding between 6′-NH 2 and 16S rRNA for antibacterial activity.

Inhibitors of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] identified by in silico molecular docking

Bioorganic & Medicinal Chemistry Letters, 2013

AAC(6)-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in-silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6)-Ib and exhibited competitive inhibition against kanamycin A.

Domain−Domain Interactions in the Aminoglycoside Antibiotic Resistance Enzyme AAC(6‘)-APH(2‘‘) †

Biochemistry, 2004

The most common determinant of aminoglycoside antibiotic resistance in Gram positive bacterial pathogens, such as Staphylococcus aureus, is a modifying enzyme, AAC(6′)-APH(2′′), capable of acetylating and phosphorylating a wide range of antibiotics. This enzyme is unique in that it is composed of two separable modification domains, and although a number of studies have been conducted on the acetyltransferase and phosphotransferase activities in isolation, little is known about the role and impact of domain interactions on antibiotic resistance. Kinetic analysis and in vivo assessment of a number of Nand C-terminal truncated proteins have demonstrated that the two domains operate independently and do not accentuate one another's resistance activity. However, the two domains are structurally integrated, and mutational analysis has demonstrated that a predicted connecting R-helix is especially critical for maintaining proper structure and function of both activities. AAC(6′)-APH(2′′) detoxifies a staggering array of aminoglycosides, where one or both activities make important contributions depending on the antibiotic. Thus, to overcome antibiotic resistance associated with AAC(6′)-APH(2′′), aminoglycosides resistant to modification and/or inhibitors against both activities must be employed. Domain-domain interactions in AAC(6′)-APH(2′′) offer a unique target for inhibitor strategies, as we show that their disruption simultaneously inhibits both activities >90%.

A Simple Structural-Based Approach to Prevent Aminoglycoside Inactivation by Bacterial Defense Proteins. Conformational Restriction Provides Effective Protection against Neomycin-B Nucleotidylation by ANT4

Journal of the American Chemical Society, 2005

The Molecular Basis of the Expansive Substrate Specificity of the Antibiotic Resistance Enzyme Aminoglycoside Acetyltransferase-6′-Aminoglycoside Phosphotransferase-2“

Journal of Biological Chemistry, 2003

The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6-aminoglycoside phosphotransferase-2؆ (AAC(6)aminoglycoside phosphotransferase-2؆, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6)-Ie, is the only member of the large AAC(6) subclass known to modify fortimicin A and catalyze Oacetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6)-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6)-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.

Broad-Spectrum Peptide Inhibitors of Aminoglycoside Antibiotic Resistance Enzymes

Chemistry & Biology, 2003

Antimicrobial Research Centre the large number and differing regiospecificities of group transfer presented by aminoglycoside modifying Department of Biochemistry McMaster University enzymes virtually guarantees that a new aminoglycoside will not be effective against all resistance mechanisms. 1200 Main Street West Hamilton, Ontario L8N 3Z5 Broad-spectrum inhibitors directed against more than one class of modifying enzyme would therefore be highly Canada 2 Department of Microbiology and Immunology desirable and would allow the rescue of aminoglycoside antibiotic activity, analogous to the employment of University of British Columbia 6174 University Boulevard ␤-lactamase inhibitors to overcome penicillin resistance [3]. Vancouver, British Columbia V6T 1Z3 Canada

Cloning, Overexpression, and Purification of Aminoglycoside Antibiotic 3-Acetyltransferase-IIIb: Conformational Studies with Bound Substrates †

Biochemistry Usa, 2002

Aminoglycoside 3-acetyltransferase-IIIb (AAC3), which acetylates N 3 amine of aminoglycoside antibiotics, was cloned from P. Aeruginosa and purified from overexpressing E. coli BL21 (DE3) cells. Bound conformations of kanamycin A and ribostamycin, in the active site of the enzyme that modifies the essential N 3B of aminoglycoside antibiotics, were determined by NMR spectroscopy. Experimentally determined interproton distances were used in a simulated annealing protocol to determine enzyme-bound conformations of both antibiotics. Two conformations, consistent with the NOE restraints, were determined for ribostamycin. The only difference between the two conformers was the orientation of the A ring with respect to the rest of the molecule. The average glycosidic dihedral angles were Φ 1A ) -22°( 3 and Ψ 1A ) -42°( 1 (conformer 1) and Φ 1A ) -67°( 0.7 and Φ 1A ) -59°( 0.8 (conformer 2). Three conformers were determined for the enzyme-bound kanamycin A. Two conformers of kanamycin A were matched well with the two conformers of ribostamycin when the A and the B rings of the antibiotics were superimposed. Conformations of kanamycin A and ribostamycin were compared to those of other aminoglycosides that are bound to different enzymes and RNA. The results lend further support to our earlier hypothesis that the A and B rings of aminoglycosides adopt a conformation that is recognized not only by the aminoglycoside-modifying enzymes but also by RNA (Serpersu, E. H., Cox, J. R., Digiammarino, E. L., Mohler, M. L., Akal, A., Ekman, D. R., and Owston, M. (2000) Cell Biochem. Biophys. 33,[309][310][311][312][313][314][315][316][317][318][319][320][321]. These results may be useful in designing new antibiotics to combat the antibiotic resistance against infectious diseases.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2")-Ia

Mechanistic Characterization of the Bifunctional Aminoglycoside-Modifying Enzyme AAC(3)-Ib/AAC(6‘)-Ib‘ from Pseudomonas aeruginosa

Biochemistry, 2007

A recently discovered bifunctional antibiotic-resistance enzyme named AAC(3)-Ib/AAC(6′)-Ib′, from Pseudomonas aeruginosa, catalyzes acetylation of aminoglycoside antibiotics. Since both domains are acetyltransferases, each was cloned and purified for mechanistic studies. The AAC(3)-Ib domain appears to be highly specific to fortimicin A and gentamicin as substrates, while the AAC(6′)-Ib′ domain exhibits a broad substrate spectrum. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition and solvent-isotope effect reveals that both domains catalyze their reactions by a steady-state ordered Bi-Bi kinetic mechanism, in which acetyl-CoA is the first substrate that binds to the active site, followed by binding of the aminoglycoside antibiotic. Subsequent to the transfer of the acetyl group, acetylated aminoglycoside is released prior to coenzyme A. The merger of two genes to create a bifunctional enzyme with expanded substrate profile would appear to be a recent trend in evolution of resistance to aminoglycoside antibiotics, of which four examples have been documented in the past few years.