Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes

Abstract

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6’)-Im

“…The AAC(6’)-I enzymes can be divided into three sub-families, based upon their structural and kinetic properties 15 16 . The AAC(6’)-Ie and AAC(6’)-Ib enzymes are classified together in sub-family C, whose members are all monomeric enzymes.…”

“…The AAC(6’)-Ig, AAC(6’)-Ih and AAC(6’)-Iy enzymes are in sub-family A and are domain-swapped dimers, with the C-terminal β-strand from one protein chain inserted between two strands from the neighboring molecule. The close proximity of the two protein chains in the dimer brings each monomer close enough such that a loop from one monomer forms part of the other chain’s active site, and vice versa 16 . The AAC(6’)-Ii enzyme is currently the sole member of sub-family B, and although this enzyme is also dimeric, the oligomer formed is different from the subfamily A dimer.…”

Insights into the Function of the N -Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis

“…5, asterisks under red highlight) that led to variants with decreased affinity for streptothricin ( Table 2), suggesting that this area is part of the structure where SatA binds streptothricin. Recently, Stogios et al showed that the antibiotic tobramycin binds to 6=-N-acetyltransferase [AAC(6=)-Ib] in an open cleft (30), highlighting the possibility that streptothricin binds to this region of SatA. We introduced substitutions at positions Y149, F154, and Y164 using the B. anthracis satA ϩ allele by site-directed mutagenesis.…”

“…Compared to our understanding of AcCoA binding, how GNATs bind their cosubstrate is less known. Recently, Stogios et al conducted an in-depth study of how the GNAT AAC(6=)-Ig binds aminoglycosides (30). In the structure of the AAC(6=)-Ig/tobramycin complex, nine residues were found to bind the antibiotic.…”

Protein structure from experimental evolution

“…3B). Proteins from the same subfamily of aminoglycoside acetyltransferases as AAC6 exist as homodimers, with the C-terminal β-strand of one protein chain inserted between two strands of the other chain ( 26 ) . We also discern these dimer interactions among the top-ranked inferred residue-residue contacts (Fig.…”