Structure of the antibiotic resistance factor spectinomycin phosphotransferase from Legionella pneumophila.
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.
Fong
Desiree H
DH
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6.
Lemke
Christopher T
CT
Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
Hwang
Jiyoung
J
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6.
Xiong
Bing
B
Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
Berghuis
Albert M
AM
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6; Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada. Electronic address: albert.berghuis@mcgill.ca.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D004352:Drug Resistance, Microbial; D007700:Kinetics; D016952:Legionella pneumophila / Q000201:enzymology*; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008024:Ligands; D020033:Protein Isoforms; D017853:Phosphotransferases (Alcohol Group Acceptor); C106858:spectinomycin phosphotransferase