Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id,a streptomycin-inactivating enzyme |
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Authors: | Meseret Ashenafi Tatiana Ammosova Sergei Nekhai W Malcolm Byrnes |
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Institution: | 1. Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA 2. Center for Sickle Cell Disease and Department of Medicine, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA
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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 32P-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. |
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