Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant

Abstract

Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.

Figure 1

Initial velocity pattern and dead-end inhibition of AAC(6′)-Ib-cr. (A) Initial velocity pattern. The symbols are experimentally determined values, while the lines are fits of the data to eq 2. (B) Inhibition pattern of lividomycin A versus ciprofloxacin at a fixed saturating concentration of AcCoA. (C) Inhibition pattern of lividomycin A versus AcCoA at a fixed saturating fixed saturating concentration of ciprofloxacin. Symbols are experimentally determined values while the lines are fits of the data to eqs 3 and 4, respectively.

Figure 2

Solvent kinetic isotope effect for AAC(6′)-Ib-cr. The symbols are experimentally determined values in H₂O (●) or 95% D₂O (■); the lines are fits of the data to eq 6. Kanamycin B and ciprofloxacin were variable substrates at a fixed, saturating concentration of AcCoA.

Figure 3

Structure of AAC(6′)-Ib-wt. Ribbon diagram of AAC(6′)-Ib-wt in complex with AcCoA and PAR.

Figure 4

F_o − F_c randomized omit map for bound aminoglycoside and donor moiety (S–H or S–COCH₃) of CoA contoured at 2.5σ for the (A) KANC/(AcCoA), (B) RIB/(CoA), and (C) PAR/(AcCoA) complexes. Prior to calculation of the electron density map temperature factors were set to 20 Ų, random errors were added to the coordinates to yield an rmsd of 0.25 Å to the parent structure, the ligands were removed, and the resulting structure was refined for 10 cycles in REFMAC. (D) Superposition of the KANC/AcCoA, RIB/CoA, and PAR/AcCoA complexes with AAC(6′)-Ib. Molecular surface and ribbon diagram of AAC(6′)-Ib are shown in gray and black, respectively. (E) Electrostatic potential of AAC(6′)-Ib mapped on the molecular surface contoured at ±5 kT (red, negative; blue, positive). PAR and AcCoA shown as sticks and colored with blue and yellow carbons, respectively.

Figure 5

Proposed mechanism of AAC(6′)-Ib-wt. (A) Stereo diagram of residues within 5 Å of aminoglycoside and AcCoA in the AAC(6′)-Ib-wt–PAR–AcCoA complex. Protein residues are shown with yellow carbons and PAR and AcCoA with green and magenta carbons, respectively. Interactions of the 6′-OH with Asp115 and the acetyl carbon of AcCoA are shown as dotted black lines. (B) Schematic of the AAC(6′)-Ib-wt enzyme mechanism with direct attack of the aminoglycoside amine on the re face of the acetyl group of AcCoA and formation of a tetrahedral intermediate.

Figure 6

Stereo diagram illustrating the model of the interaction of (A) KANC and (B) CIP with the Asp179Tyr/Trp102Arg–AAC(6′)-Ib-cr mutant, illustrating the interaction of the acceptor with the acetyl group of AcCoA and Asp115.