Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.

The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.

The Gram-positive pathogen Enterococcus faecium is intrinsically resistant to aminoglycoside antibiotics due to the presence of a chromosomally encoded aminoglycoside 6'-N-acetyltransferase [AAC(6')-Ii]. This enzyme is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily and is therefore structurally homologous to proteins that catalyze acetyl transfer to diverse acyl-accepting substrates. This study reports the investigation of several potential catalytic residues that are present in the AAC(6')-Ii active site and also conserved in many GNAT enzymes. Site-directed mutagenesis of Glu72, His74, Leu76, and Tyr147 with characterization of the purified site mutants gave valuable information about the roles of these amino acids in acetyl transfer chemistry. More specifically, steady-state kinetic analysis of protein activity, solvent viscosity effects, pH studies, and antibiotic resistance profiles were all used to assess the roles of Glu72 and His74 as potential active site bases, Tyr147 as a general acid, and the importance of the amide NH group of Leu76 in transition-state stabilization. Taken together, our results indicate that Glu72 is not involved in general base catalysis, but is instead critical for the proper positioning and orientation of aminoglycoside substrates in the active site. Similarly, His74 is also not acting as the active site base, with pH studies revealing that this residue must be protonated for optimal AAC(6')-Ii activity. Mutation of Tyr147 was found not to affect the chemical step of catalysis, and our results were not consistent with this residue acting as a general acid. Last, the amide NH group of Leu76 is implicated in important interactions with acetyl-CoA and transition-state stabilization. In summary, the work described here provides important information regarding the molecular mechanism of AAC(6')-Ii catalysis that allows us to contrast our findings with those of other GNAT proteins and to demonstrate that these enzymes use a variety of chemical mechanisms to accelerate acyl transfer.

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.