title: Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme.
abstract: As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of (32)P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K(m)(streptomycin) of 0.38 ± 0.13 mM, K(m)(ATP) of 1.03 ± 0.1 mM, V(max) of 3.2 ± 1.1 μmol/min/mg, and k(cat) of 1.7 ± 0.6 s(-1). Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA.
authors.forename: Meseret
authors.initials: M
authors.lastname: Ashenafi
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ammosova
authors.forename: Sergei
authors.initials: S
authors.lastname: Nekhai
authors.forename: W Malcolm
authors.initials: WM
authors.lastname: Byrnes
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology; D013307:Streptomycin / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C022680:streptomycin 6-kinase; D013307:Streptomycin