Crystal structure of an aminoglycoside 6′-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold

Abstract

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.

Evolution of insect arylalkylamine N -acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti

“…The tunnel thus created in the center of the β-sheet is necessary for the binding of the CoA cofactor and the substrate in other GCN5-related NAT (GNAT) proteins (27). All three proteins have four conserved sequence motifs, termed C, D, A, and B in N-to C-terminal order, and contain a common fold core that includes α-helices on both sides of six-stranded mixed β-sheets (β1-β6) of the GNAT superfamily (28) (Fig. 2).…”

“…In addition, paaNAT7 has one more extra β strand, β0, at the N-terminus. Although GNAT superfamily members have four conserved sequence motifs (34) and contain a common fold core (28), they all display limited sequence similarity and retain few invariant residues (27), particularly in the regions where a more inconsistent stock of structures is built around the common core. This makes homology detection a challenge for GNAT superfamily members using sequence comparison alone (27,35).…”

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

“…However, assays were scaled down from 1-ml to 250-l volumes, so they could be conducted in 96-well microtiter plates using a Molecular Devices SpectraMax Plus microtiter plate reader. For more sensitive assays, acetyltransferase activity was determined using [1-14 C]acetylCoA and a phosphocellulose binding assay described previously (16 For the solvent viscosity effect experiments, steady-state kinetic parameters were determined with varying concentrations of the microviscosogen glycerol (0, 15, 22.5, and 30% (w/v)). The viscosity of the solutions was determined using an Ostwald viscometer in triplicate, and the slope of a plot of relative viscosity versus rate o /rate viscogen reveals the solvent viscosity effect (SVE).…”

“…In the histone acetyltransferase tGCN5, Glu-173 has been suggested to fulfill this role (30,31), whereas in AANAT, the active site base appears to be His-122 (29). The three-dimensional structure of AAC(6Ј)-APH(2Љ) has not been determined, so we used available sequence alignments (16,40) to generate a partial alignment of GNAT family members that could aid in identifying the putative active site base in AAC(6Ј)-Ie (Fig. 4).…”

“…Glu-173 has been suggested to be the active site base in tGCN5 (30,31), and His-122 has been suggested to be the active site base in AANAT (29). The alignment is based on previous structurebased alignments (16,40). AAC(6Ј)-Ie.…”