Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase.

Yang Chen,Joakim Näsvall,Dan I Andersson,Shiying Wu,Maria Selmer

doi:10.1107/s1399004715016429

Abstract

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3'')(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

Highlights

Ever since the discovery of the first aminoglycoside antibiotic, streptomycin (Schatz et al, 1944), which was isolated from Streptomyces griseus, these broad-spectrum antibiotics have been widely used in the treatment of bacterial infections
We have presented the first crystal structure of an aminoglycoside O-nucleotidyltransferases (ANTs)(300)(9) adenyltransferase: AadA from S. enterica
We have shown using Isothermal titration calorimetry (ITC) that ATP binds to AadA before the aminoglycoside substrate and positions the two domains for aminoglycoside binding in the interdomain cleft

Summary

Introduction

Ever since the discovery of the first aminoglycoside antibiotic, streptomycin (Schatz et al, 1944), which was isolated from Streptomyces griseus, these broad-spectrum antibiotics have been widely used in the treatment of bacterial infections. Crystal structures of aminoglycosides bound to the 30S ribosomal subunit (Brodersen et al, 2000; Carter et al, 2000; Demirci et al, 2013) shed new light on their mechanisms of increasing errors in decoding. The ANT enzymes in general use ATP and magnesium to adenylate specific hydroxyl groups of their substrates, while some of them can use other NTPs and/or other divalent ions. They are further classified depending on the site of substrate modification (Azucena & Mobashery, 2001; Jana & Deb, 2006), which is linked to their substrate specificity. Crystal structures are available of the ANT(40)-Ia kanamycin nucleotidyltransferase (PDB entry 1kny; Pedersen et al, 1995; Sakon et al, 1993), the ANT(40)-IIb enzyme (PDB entries 4ebj and 4ebk; Center for Structural Genomics of Infectious Diseases, unpublished work), the ANT(6)-Ia enzyme (PDB entry 2pbe; New York SGX Research Center for Structural Genomics, unpublished work) and, recently, the ANT(200)-Ia enzyme (PDB entries 4wqk and 4wql; Cox et al, 2015)

Methods

Results

Conclusion