Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Abstract

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3) isozymes and an APH(2؆) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3) and APH(2؆) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.Aminoglycosides are a class of commonly used broadspectrum antibiotics that target the bacterial ribosome. They are characterized by their signature chemical structure, an aminocyclitol nucleus. These antibiotics can be further categorized into two groups: the first group includes those that contain a 2-deoxystreptamine core, such as kanamycin, and the second, smaller group includes those that contain a non-2-deoxystreptamine core (Fig. 1A). Spectinomycin has a streptamine core and it is used in the treatment of acute gonococcal infections (1). Unfortunately, the efficacy of aminoglycosides has been compromised due to the continuous rise of drug resistance in pathogens. Resistance to aminoglycosides can be attributed to several mechanisms, of which enzymatic inactivation of the aminoglycoside is the most prevalent in the clinical setting. Aminoglycosides can be inactivated by the addition of an acetyl, a nucleotidyl, or a phosphate group by acetyltransferases, nucleotidyltransferases, or phosphotransferases (kinases; APHs), 5 respectively (2). Nomenclature of aminoglycoside-modifying enzymes follows the convention proposed by Shaw et al. (2): the type of modifying enzyme is identified by acetyltransferase, nucleotidyltransferase, or APH; this is followed by, in parentheses, the enzyme regiospecificity; then the substrate profile is designated by a roman numeral, and the unique amino acid sequence is denoted by a small letter.APHs generally yield high levels of resista...

Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

“…Overall, it appears that AAC(6=)-Ie/ APH(2Љ)-Ia maintains a rigid conformation while binding substrate. This behavior is similar to what has been observed for APH(3=)-IIIa, another clinically important aminoglycoside resistance enzyme (6), but is in sharp contrast to what is seen for APH(9)-Ia, an enzyme whose role in antibiotic resistance is debatable (17). We have previously suggested that the absence of major conformational changes may signify that the aminoglycoside resistance enzyme is optimized for rapid drug detoxification and, thus, is an important feature of dedicated resistance enzymes (6).…”

Structural Basis for Dual Nucleotide Selectivity of Aminoglycoside 2″-Phosphotransferase IVa Provides Insight on Determinants of Nucleotide Specificity of Aminoglycoside Kinases

“…Taken together, the inability to form an ordered solvent network and the lack of sufficient hydrogen-bonding interactions due to an altered conformation of the linker loop act synergistically to prohibit APH(3Ј)-IIIa from binding GTP, and neither contributing factor is controlled by individual amino acids. That other ATP-selective APHs cannot bind GTP for similar reasons is substantiated by comparisons with available structural data, such as the nucleotide-bound structure of APH(9)-Ia (11).…”

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

“…9,10 Aminoglycoside phosphotransferases (APHs) modify the 2-deoxystreptamine antibiotics by phosphorylation of their hydroxyl groups at Positions 4, 6, 3 0 , 2 00 , 3 00 , 7 00 (Fig. 1), or at Position 9 of spectinomycin 11 ). The enzymes are named according to the reaction they catalyze, the position on the aminoglycoside that they modify, their resistance profile (which nucleotides and aminoglycosides they will bind) indicated by a Roman numeral, and their genetic variability indicated by a lower-case letter.…”

“…The ATP-binding site is sandwiched between the N-terminal and C-terminal domains, with the aminoglycoside substrate-binding site located in a cleft in the C-terminal domain. There are three additional aminoglycoside phosphotransferase structures, APH(9) (spectinomycin phosphotransferase) 11 and two from the APH(3 0 ) family of enzymes, 19,20 and despite a very low sequence identity between these APH(9), APH(3 00 ), and APH(2 00 ) families, there is a marked degree of structural similarity. 11,18 Here, we report the X-ray structure, substrate profile, and kinetic mechanism of a second member of the APH(2 00 ) subfamily, the APH(2 00 )-IVa aminoglycoside phosphotransferase, and for the first time we report the determinants of NTP recognition by these clinically important enzymes.…”